Anti-microRNA oligonucleotide molecules

ABSTRACT

The invention relates to isolated anti-microRNA molecules. In another embodiment, the invention relates to an isolated microRNA molecule. In yet another embodiment, the invention provides a method for inhibiting microRNP activity in a cell.

The invention claimed herein was made with the help of grant number 1 R01 GM068476-01 from NIH/NIGMS. The U.S. government has certain rights in the invention.

This application is a U.S. National Phase Application of International Application No. PCT/US05/04714 filed on Feb. 11, 2005 and asserts priority to U.S. application Ser. No. 10/845,057 filed on May 13, 2004, which is a continuing application of U.S. application Ser. No. 10/778,908 filed on Feb. 13, 2004. The specifications of International Application No. PCT/US05/04714, U.S. application Ser. No. 10/845,057, and U.S. application Ser. No. 10/778,908 are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

RNA silencing is a fundamental mechanism of gene regulation that uses double-stranded RNA (dsRNA) derived 21- to 28-nucleotide (nt) small RNAs to guide mRNA degradation, control mRNA translation or chromatin modification. Recently, several hundred novel genes were identified in plants and animals that encode transcripts that contain short dsRNA hairpins.

Defined 22-nt RNAs, referred to as microRNAs (miRNAs), are reported to be excised by dsRNA specific endonucleases from the hairpin precursors. The miRNAs are incorporated into ribonucleoprotein particles (miRNPs).

Plant miRNAs target mRNAs containing sequence segments with high complementarity for degradation or suppress translation of partially complementary mRNAs. Animal miRNAs appear to act predominantly as translational repressors. However, animal miRNAs have also been reported to guide RNA degradation. This indicates that animal miRNPs act like small interfering RNA (siRNA)-induced silencing complexes (RISCs).

Understanding the biological function of miRNAs requires knowledge of their mRNA targets. Bioinformatic approaches have been used to predict mRNA targets, among which transcription factors and proapoptotic genes were prominent candidates. Processes such as Notch signaling, cell proliferation, morphogenesis and axon guidance appear to be controlled by miRNA genes.

Therefore, there is a need for materials and methods that can help elucidate the function of known and future microRNAs. Due to the ability of microRNAs to induce RNA degradation or repress translation of mRNA which encode important proteins, there is also a need for novel compositions for inhibiting microRNA-induced cleavage or repression of mRNAs.

SUMMARY THE INVENTION

In one embodiment, the invention provides an isolated single stranded anti-microRNA molecule comprising a minimum of ten moieties and a maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, each base forming a Watson-Crick base pair with a complementary base wherein at least ten contiguous bases have the same sequence as a sequence of bases in any one of the anti-microRNA molecules shown in Tables 1-4, except that up to thirty percent of the bases pairs may be wobble base pairs, and up to 10% of the contiguous bases may be additions, deletions, mismatches, or combinations thereof; no more than fifty percent of the contiguous moieties contain deoxyribonuleotide backbone units; the moiety in the molecule at the position corresponding to position 11 of the microRNA is non-complementary; and the molecule is capable of inhibiting microRNP activity.

In another embodiment, the invention provides a method for inhibiting microRNP activity in a cell, the microRNP comprising a microRNA molecule, the microRNA molecule comprising a sequences of bases complementary of the sequence of bases in a single stranded anti-microRNA molecule, the method comprising introducing into the cell the single-stranded anti-microRNA molecule comprising a sequence of a minimum of ten moieties and a maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, each base forming a Watson-Crick base pair with a complementary base, wherein at least ten contiguous bases of the anti-microRNA molecule are complementary to the microRNA, except that up to thirty percent of the bases may be substituted by wobble base pairs, and up to ten percent of the at least ten moieties may be additions, deletions, mismatches, or combinations thereof; no more than fifty percent of the contiguous moieties contain deoxyribonuleotide backbone units; and the moiety in the molecule at the position corresponding to position 11 of the microRNA is non-complementary.

In another embodiment, the invention provides an isolated microRNA molecule comprising a minimum of ten moieties and a maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, wherein at least ten contiguous bases have the same sequence as a sequence of bases in any one of the microRNA molecules shown in Table 2, except that up to thirty percent of the bases pairs may be wobble base pairs, and up to 10% of the contiguous bases may be additions, deletions, mismatches, or combinations thereof; and no more than fifty percent of the contiguous moieties contain deoxyribonuleotide backbone units.

In another embodiment, the invention provides an isolated microRNA molecule comprising a minimum of ten moieties and a maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, wherein at least ten contiguous bases have any one of the microRNA sequences shown in Tables 1, 3 and 4, except that up to thirty percent of the bases pairs may be wobble base pairs, and up to 10% of the contiguous bases may be additions, deletions, mismatches, or combinations thereof; no more than fifty percent of the contiguous moieties contain deoxyribonuleotide backbone units; and is modified for increased nuclease resistance.

In yet another embodiment, the invention provides an isolated single stranded anti-microRNA molecule comprising a minimum of ten moieties and a maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, each base forming a Watson-Crick base pair with a complementary base wherein at least ten contiguous bases have the same sequence as a sequence of bases in any one of the anti-microRNA molecules shown in Tables 1-4, except that up to thirty percent of the bases pairs may be wobble base pairs, and up to 10% of the contiguous bases may be additions; deletions, mismatches, or combinations thereof; no more than fifty percent of the contiguous moieties contain deoxyribonuleotide backbone units; and the molecule is capable of inhibiting microRNP activity.

In yet a further embodiment, the invention provides a method for inhibiting microRNP activity in a cell, the microRNP comprising a microRNA molecule, the microRNA molecule comprising a sequences of bases complementary of the sequence of bases in a single stranded anti-microRNA molecule, the method comprising introducing into the cell the single-stranded anti-microRNA molecule comprising a sequence of a minimum of ten moieties and a maximum of fifty moieties on a molecular backbone, the molecular backbone comprising backbone units, each moiety comprising a base bonded to a backbone unit, each base forming a Watson-Crick base pair with a complementary base, wherein at least ten contiguous bases of the anti-microRNA molecule are complementary to the microRNA, except that up to thirty percent of the bases may be substituted by wobble base pairs, and up to ten percent of the at least ten moieties may be additions, deletions, mismatches, or combinations thereof; and no more than fifty percent of the contiguous moieties contain deoxyribonuleotide backbone units.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the modified nucleotide units discussed in the specification. B denotes any one of the following nucleic acid bases: adenosine, cytidine, guanosine, thymine, or uridine.

FIG. 2. Antisense 2′-O-methyl oligoribonucleotide specifically inhibit miR-21 guided cleavage activity in HeLa cell S100 cytoplasmic extracts. The black bar to the left of the RNase T1 ladder represents the region of the target RNA complementary to miR-21. Oligonucleotides complementary to miR-21 were pre-incubated in S100 extracts prior to the addition of ³²P-cap-labelled cleavage substrate. Cleavage bands and T1 hydrolysis bands appear as doublets after a 1-nt slipping of the T7 RNA polymerase near the middle of the transcript indicated by the asterisk.

FIG. 3. Antisense 2′-O-methyl oligoribonucleotides interfere with endogenous miR-21 RNP cleavage in HeLa cells. HeLa cells were transfected with pHcRed and pEGFP or its derivatives, with or without inhibitory or control oligonucleotides. EGFP and HcRed protein fluorescence were excited and recorded individually by fluorescence microscopy 24 h after transfection. Co-expression of co-transfected reporter plasmids was documented by superimposing of the fluorescence images in the right panel.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to an isolated single stranded anti-microRNA molecule. The molecule comprises a minimum number of ten moieties, preferably a minimum of thirteen, more preferably a minimum of fifteen, even more preferably a minimum of 18, and most preferably a minimum of 21 moieties.

The anti-microRNA molecule comprises a maximum number of fifty moieties, preferably a maximum of forty, more preferably a maximum of thirty, even more preferably a maximum of twenty-five, and most preferably a maximum of twenty-three moieties. A suitable range of minimum and maximum number of moieties may be obtained by combining any of the above minima with any of the above maxima.

Each moiety comprises a base bonded to a backbone unit. In this specification, a base refers to any one of the nucleic acid bases present in DNA or RNA. The base can be a purine or pyrimidine. Examples of purine bases include adenine (A) and guanine (G). Examples of pyrimidine bases include thymine (T), cytosine (C) and uracil (U). Each base of the moiety forms a Watson-Crick base pair with a complementary base.

Watson-Crick base pairs as used herein refers to the hydrogen bonding interaction between, for example, the following bases: adenine and thymine (A=T); adenine and uracil (A=U); and cytosine and guanine (C=G). The adenine can be replaced with 2,6-diaminopurine without compromising base-pairing.

The backbone unit may be any molecular unit that is able stably to bind to a base and to form an oligomeric chain. Suitable backbone units are well known to those in the art.

For example, suitable backbone units include sugar-phosphate groups, such as the sugar-phosphate groups present in ribonucleotides, deoxyribonucleotides, phosphorothioate deoxyribose groups, N′3-N′5 phosphoroamidate deoxyribose groups, 2′0-alkyl-ribose phosphate groups, 2′-O-alkyl-alkoxy ribose phosphate groups, ribose phosphate group containing a methylene bridge, 2′-Fluororibose phosphate groups, morpholino phosphoroamidate groups, cyclohexene groups, tricyclo phosphate groups, and amino acid molecules.

In one embodiment, the anti-microRNA molecule comprises at least one moiety which is a ribonucleotide moiety or a deoxyribonucleotide moiety.

In another embodiment, the anti-microRNA molecule comprises at least one moiety which confers increased nuclease resistance. The nuclease can be an exonuclease, an endonuclease, or both. The exonuclease can be a 3′→5′ exonuclease or a 5′→3′ exonuclease. Examples of 3′→5′ human exonuclease include PNPT1, Werner syndrome helicase, RRP40, RRP41, RRP42, RRP45, and RRP46. Examples of 5′→3′ exonuclease include XRN2, and FEN1. Examples of endonucleases include Dicer, Drosha, RNase4, Ribonuclease P, Ribonuclease H1, DHP1, ERCC-1 and OGG1. Examples of nucleases which function as both an exonuclease and an endonuclease include APE1 and EXO1.

An anti-microRNA molecule comprising at least one moiety which confers increased nuclease resistance means a sequence of moieties wherein at least one moiety is not recognized by a nuclease. Therefore, the nuclease resistance of the molecule is increased compared to a sequence containing only unmodified ribonucleotide, unmodified deoxyribonucleotide or both. Such modified moieties are well known in the art, and were reviewed, for example, by Kurreck, Eur. J. Biochem. 270, 1628-1644 (2003).

A modified moiety can occur at any position in the anti-microRNA molecule. For example, to protect the anti-microRNA molecule against 3′→5′ exonucleases, the molecule can have at least one modified moiety at the 3′ end of the molecule and preferably at least two modified moieties at the 3′ end. If it is desirable to protect the molecule against 5′→3′ exonuclease, the anti-microRNA molecule can have at least one modified moiety and preferably at least two modified moieties at the 5′ end of the molecule. The anti-microRNA molecule can also have at least one and preferably at least two modified moieties between the 5′ and 3′ end of the molecule to increase resistance of the molecule to endonucleases. In one embodiment, all of the moieties are nuclease resistant.

In another embodiment, the anti-microRNA molecule comprises at least one modified deoxyribonucleotide moiety. Suitable modified deoxyribonucleotide moieties are known in the art.

A suitable example of a modified deoxyribonucleotide moiety is a phosphorothioate deoxyribonucleotide moiety. See structure 1 in FIG. 1. An anti-microRNA molecule comprising more than one phosphorothioate deoxyribonucleotide moiety is referred to as phosphorothioate (PS) DNA. See, for example, Eckstein, Antisense Nucleic Acids Drug Dev. 10, 117-121 (2000).

Another suitable example of a modified deoxyribonucleotide moiety is an N′3-N′5 phosphoroamidate deoxyribonucleotide moiety. See structure 2 in FIG. 1. An oligonucleotide molecule comprising more than one phosphoroamidate deoxyribonucleotide moiety is referred to as phosphoroamidate (NP) DNA. See, for example, Gryaznov et al., J. Am. Chem. Soc. 116, 3143-3144 (1994).

In another embodiment, the molecule comprises at least one modified ribonucleotide moiety. Suitable modified ribonucleotide moieties are known in the art.

A suitable example of a modified ribonucleotide moiety is a ribonucleotide moiety that is substituted at the 2′ position. The substituents at the 2′ position may, for example, be a C₁ to C₄ alkyl group. The C₁ to C₄ alkyl group may be saturated or unsaturated, and unbranched or branched. Some examples of C₁ to C₄ alkyl groups include ethyl, isopropyl, and allyl. The preferred C₁ to C₄ alkyl group is methyl. See structure 3 in FIG. 1. An oligoribonucleotide molecule comprising more than one ribonucleotide moiety that is substituted at the 2′ position with a C₁ to C₄ alkyl group is referred to as a 2′-O-(C₁-C₄ alkyl) RNA, e.g., 2′-O-methyl RNA (OMe RNA).

Another suitable example of a substituent at the 2′ position of a modified ribonucleotide moiety is a C₁ to C₄ alkoxy-C₁ to C₄ alkyl group. The C₁ to C₄ alkoxy (alkyloxy) and C₁ to C₄ alkyl group may comprise any of the alkyl groups described above. The preferred C₁ to C₄ alkoxy-C₁ to C₄ alkyl group is methoxyethyl. See structure 4 in FIG. 1. An oligonucleotide molecule comprising more than one ribonucleotide moiety that is substituted at the 2′ position. with a C₁ to C₄ alkoxy-C₁ to C₄ alkyl group is referred to as a 2′-O-(C₁ to C₄ alkoxy-C₁ to C₄ alkyl) RNA, e.g., 2′-O-methoxyethyl RNA (MOE RNA).

Another suitable example of a modified ribonucleotide moiety is a ribonucleotide that has a methylene bridge between the 2′-oxygen atom and the 4′-carbon atom. See structure 5 in FIG. 1. An oligoribonucleotide molecule comprising more than one ribonucleotide moiety that has a methylene bridge between the 2′-oxygen atom and the 4′-carbon atom is referred to as locked nucleic acid (LNA). See, for example, Kurreck et al., Nucleic Acids Res. 30, 1911-1918 (2002); Elayadi et al., Curr. Opinion Invest. Drugs 2, 558-561 (2001); Ørum et al., Curr. Opinion Mol. Ther. 3, 239-243 (2001); Koshkin et al., Tetrahedron 54, 3607-3630 (1998); Obika et al., Tetrahedron Lett.39, 5401-5404 (1998). Locked nucleic acids are commercially available from Proligo (Paris, France and Boulder, Colo., USA).

Another suitable example of a modified ribonucleotide moiety is a ribonucleotide that is substituted at the 2′ position with fluoro group. A modified ribonucleotide moiety having a fluoro group at the 2′ position is a 2′-fluororibonucleotide moiety. Such moieties are known in the art. Molecules comprising more than one 2′-fluororibonucleotide moiety are referred to herein as 2′-fluororibo nucleic acids (FANA). See structure 7 in FIG. 1. Damha et al., J. Am. Chem. Soc. 120, 12976-12977 (1998).

In another embodiment, the anti-microRNA molecule comprises at least one base bonded to an amino acid residue. Moieties that have at least one base bonded to an amino acid residue will be referred to herein as peptide nucleic acid (PNA) moieties. Such moieties are nuclease resistance, and are known in the art. Molecules having more than one PNA moiety are referred to as peptide nucleic acids. See structure 6 in FIG. 1. Nielson, Methods Enzymol. 313, 156-164 (1999); Elayadi, et al, id.; Braasch et al., Biochemistry 41, 4503-4509 (2002), Nielsen et al., Science 254, 1497-1500 (1991).

The amino acids can be any amino acid, including natural or non-natural amino acids. Naturally occurring amino acids include, for example, the twenty most common amino acids normally found in proteins, i.e., alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Glu), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ileu), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser, threonine (Thr), tryptophan, (Trp), tyrosine (Tyr), and valine (Val).

The non-natural amino acids may, for example, comprise alkyl, aryl, or alkylaryl groups. Some examples of alkyl amino acids include α-aminobutyric acid, β-aminobutyric acid, γ-aminobutyric acid, δ-aminovaleric acid, and ε-aminocaproic acid. Some examples of aryl amino acids include ortho-, meta, and para-aminobenzoic acid. Some examples of alkylaryl amino acids include ortho-, meta-, and para-aminophenylacetic acid, and γ-phenyl-β-aminobutyric acid.

Non-naturally occurring amino acids also include derivatives of naturally occurring amino acids. The derivative of a naturally occurring amino acid may, for example, include the addition or one or more chemical groups to the naturally occurring amino acid.

For example, one or more chemical groups can be added to one or more of the 2′, 3′, 4′, 5′, or 6′ position of the aromatic ring of a phenylalanine or tyrosine residue, or the 4′, 5′, 6′, or 7′ position of the benzo ring of a tryptophan residue. The group can be any chemical group that can be added to an aromatic ring. Some examples of such groups include hydroxyl, C₁-C₄ alkoxy, amino, methylamino, dimethylamino, nitro, halo (i.e., fluoro, chloro, bromo, or iodo), or branched or unbranched C₁-C₄ alkyl, such as methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, or t-butyl.

Furthermore, other examples of non-naturally occurring amino acids which are derivatives of naturally occurring amino acids include norvaline (Nva), norleucine (Nle), and hydroxyproline (Hyp).

The amino acids can be identical or different from one another. Bases are attached to the amino acid unit by molecular linkages. Examples of linkages are methylene carbonyl, ethylene carbonyl and ethyl linkages. (Nielsen et al., Peptide Nucleic Acids-Protocols and Applications, Horizon Scientific Press, pages 1-19, Nielsen et al., Science 254: 1497-1500.)

One example of a PNA moiety is N-(2-aminoethyl)-glycine. Further examples of PNA moieties include cyclohexyl PNA, retro-inverso, phosphone, propionyl and aminoproline PNA.

PNA can be, chemically synthesized by methods known in the art, e.g. by modified Fmoc or tBoc peptide synthesis protocols. The PNA has many desirable properties, including high melting temperatures (Tm), high base-pairing specificity with nucleic acid and an uncharged molecular backbone. Additionally, the PNA does not confer RNase H sensitivity on the target RNA, and generally has good metabolic stability.

Peptide nucleic acids are also commercially available from Applied Biosystems (Foster City, Calif., USA).

In another embodiment, the anti-microRNA molecule comprises at least one morpholino phosphoroamidate nucleotide moiety. A morpholino phosphoroamidate nucleotide moiety is a modified moiety which is nuclease resistant. Such moieties are known in the art. Molecules comprising more than one morpholino phosphoroamidate nucleotide moiety are referred to as morpholino (MF) nucleic acids. See structure 8 in FIG. 1. Heasman, Dev. Biol. 243, 209-214 (2002). Morpholono oligonucleotides are commercially available from Gene Tools LLC (Corvallis, Oreg., USA).

In another embodiment, the anti-microRNA molecule comprises at least one cyclohexene nucleotide moiety. A cyclohexene nucleotide moiety is a modified moiety which is nuclease resistant. Such moieties are known in the art. Molecules comprising more than one cyclohexene nucleotide moiety are referred to as cyclohexene nucleic acids (CeNA). See structure 10 in FIG. 1. Wang et al., J. Am. Chem. Soc. 122, 8595-8602 (2000), Verbeure et al., Nucleic Acids Res. 29, 4941-4947 (2001).

In another embodiment, the anti-microRNA molecule comprises at least one tricyclo nucleotide moiety. A tricyclo nucleotide moiety is a modified moiety which is nuclease resistant. Such moieties are known in the art. Steffens et al., J. Am. Chem. Soc. 119,-11548-11549 (1-997), Renneberg et al., J. Am. Chem. Soc. 124, 5993-6002 (2002). Molecules comprising more than one tricyclo nucleotide moiety are referred to as tricyclo nucleic acids (tcDNA). See structure 9 in FIG. 1.

In another embodiment, to increase nuclease resistance of the anti-microRNA molecules of the present invention to exonucleases, inverted nucleotide caps can be attached to the 5′. end, the 3′ end, or both ends of the molecule. An inverted nucleotide cap refers to a 3′→5′ sequence of nucleic acids attached to the anti-microRNA molecule at the 5′ and/or the 3′ end. There is no limit to the maximum number of nucleotides in the inverted cap just as long as it does not interfere with binding of the anti-microRNA molecule to its target microRNA. Any nucleotide can be used in the inverted nucleotide cap. Typically, the inverted nucleotide cap is one nucleotide in length. The nucleotide for the inverted cap is generally thymine, but can be any nucleotide such as adenine, guanine, uracil, or cytosine.

Alternatively, an ethylene glycol compound and/or amino linkers can be attached to the either or both ends of the anti-microRNA molecule. Amino linkers can also be used to increase nuclease resistance of the anti-microRNA molecules to endonucleases. The table below lists some examples of amino linkers. The below listed amino linker are commercially available from TriLink Biotechnologies, San Diego, Calif.

2′-Deoxycytidine-5-C6 Amino Linker (3′ Terminus) 2′-Deoxycytidine-5-C6 Amino Linker (5′ or Internal) 3′ C3 Amino Linker 3′ C6 Amino Linker 3′ C7 Amino Linker 5′ C12 Amino Linker 5′ C3 Amino Linker 5′ C6 Amino Linker C7 Internal Amino Linker Thymidine-5-C2 Amino Linker (5′ or Internal) Thymidine-5-C6 Amino Linker (3′ Terminus) Thymidine-5-C6 Amino Linker (Internal)

Chimeric anti-microRNA molecules containing a mixture of any of the moieties mentioned above are also known, and may be made by methods known, in the art. See, for example, references cited above, and Wang et al, Proc. Natl. Acad. Sci. USA 96,13989-13994 (1999), Liang et al., Eur. J. Biochem. 269, 5753-5758 (2002), Lok et al., Biochemistry 41, 3457-3467 (2002), and Damha et al., J. Am. Chem. Soc. 120, 12976-12977 (2002).

The molecules of the invention comprise at least ten contiguous, preferably at least thirteen contiguous, more preferably at least fifteen contiguous, and even more preferably at least twenty contiguous bases that have the same sequence as a sequence of bases in any one of the anti-microRNA molecules shown in Tables 1-4. The anti-microRNA molecules optimally comprise the entire sequence of any one of the anti-microRNA molecule sequences shown in Tables 1-4.

For the contiguous bases mentioned above, up to thirty percent of the base pairs may be substituted by wobble base pairs. As used herein, wobble base pairs refers to either: i) substitution of a cytosine with a uracil, or 2) the substitution of a adenine with a guanine, in the sequence of the anti-microRNA molecule. These wobble base pairs are generally referred to as UG or GU wobbles. Below is a table showing the number of contiguous bases and the maximum number of wobble base pairs in the anti-microRNA molecule:

Table for Number of Wobble Bases No. of Contiguous Bases 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Max. No. of Wobble 3 3 3 3 4 4 4 5 5 5 6 6 6 6 Base Pairs

Further, up to ten percent, and preferably up to five percent of the contiguous bases can be additions, deletions, mismatches or combinations thereof Additions refer to the insertion in the contiguous sequence of any moiety described above comprising any one of the bases described above. Deletions refer to the removal of any moiety present in the contiguous sequence. Mismatches refer to the substitution of one of the moieties comprising a base in the contiguous sequence with any of the above described moieties comprising a different base.

The additions, deletions or mismatches can occur anywhere in the contiguous sequence, for example, at either end of the contiguous sequence or within the contiguous sequence of the anti-microRNA molecule. If the contiguous sequence is relatively short, such as from about ten to about 15 moieties in length, preferably the additions, deletions or mismatches occur at the end of the contiguous sequence. If the contiguous sequence is relatively long, such as a minimum of sixteen contiguous sequences, then the additions, deletions, or mismatches can occur anywhere in the contiguous sequence. Below is a table showing the number of contiguous bases and the maximum number of additions, deletions, mismatches or combinations thereof:

Table for Up to 10% No. of Contiguous Bases 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Max. No. of Additions, 1 1 1 1 1 1 1 1 1 1 2 2 2 2 Deletions and/or Mismatches

Table for Up to 5% No. of Contiguous Bases 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Max. No. of Additions, 0 0 0 0 0 0 0 0 0 0 1 1 1 1 Deletions and/or Mismatches

Furthermore, no more than fifty percent, and preferably no more than thirty percent, of the contiguous moieties contain deoxyribonucleotide backbone units. Below is a table showing the number of contiguous bases and the maximum number of deoxyribonucleotide backbone units:

Table for Fifty Percent Deoxyribonucleotide Backbone Units No. of Contiguous Bases 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Max. No. of 5 5 6 6 7 7 8 8 9 9 10 10 11 11 Deoxyribonucleotide Backbone Units

Table for Thirty Percent Deoxyribonucleotide Backbone Units No. of Contiguous Bases 10 11 12 13 14 15 16 17 18 19 20 21 22 23 Max. No. of 3 3 3 3 4 4 4 5 5 5 6 6 6 6 Deoxyribonucleotide Backbone Units

The moiety in the anti-RNA molecule at the position corresponding to position 11 of the microRNA is optionally non-complementary to a microRNA. The moiety in the anti-microRNA molecule corresponding to position 11 of the microRNA can be rendered non-complementary by an addition, deletion or mismatch as described above.

In another embodiment, if the anti-microRNA molecule comprises only unmodified moieties, then the anti-microRNA molecules comprises at least one base, in the at least ten contiguous bases, which is non-complementary to the microRNA and/or comprises an inverted nucleotide cap, ethylene glycol compound or an amino linker.

In yet another embodiment, if the at least ten contiguous bases in an anti-microRNA molecule is perfectly (i.e., 100%) complementary to ten contiguous bases in a microRNA, then the anti-microRNA molecule contains at least one modified moiety in the at least ten contiguous bases and/or comprises an inverted nucleotide cap, ethylene glycol compound or an amino linker.

As stated above, the maximum length of the anti-microRNA molecule is 50 moieties. Any number of moieties having any base sequence can be added to the contiguous base sequence. The additional moieties can be added to the 5′ end, the 3′ end, or to both ends of the contiguous sequence.

MicroRNA molecules are derived from genomic loci and are produced from specific microRNA genes. Mature microRNA molecules are processed from precursor transcripts that form local hairpin structures. The hairpin structures are typically cleaved by an enzyme known as Dicer, which generates one microRNA duplex. See Bartel, Cell 116, 281-297 (2004) for a review on microRNA molecules. The article by Bartel is hereby incorporated by reference.

Each strand of a microRNA is packaged in a microRNA ribonucleoprotein complex (microRNP). A microRNP in, for example, humans, also includes the proteins eIF2C2, the helicase Gemin3, and Gemin 4.

The sequence of bases in the anti-microRNA molecules of the present invention can be derived from a microRNA from any species e.g. such as a fly (e.g., Drosophila melanogaster), a worm (e.g., C. elegans). Preferably the sequence of bases is found in mammals, especially humans (H. sapiens), mice (e.g., M. musculus), and rats (R. norvegicus).

The anti-microRNA molecule is preferably isolated, which means that it is essentially free of other nucleic acids. Essentially free from other nucleic acids means that it is at least 90%, preferably at least 95% and, more preferably, at least 98% free of other nucleic acids.

Preferably, the molecule is essentially pure, which means that the molecules is free not only of other nucleic acids, but also of other materials used in the synthesis of the molecule, such as, for example, enzymes used in the synthesis of the molecule. The molecule is at least 90% free, preferably at least 95% free and, more preferably, at least 98% free of such materials.

The anti-microRNA molecules of the present invention are capable of inhibiting microRNP activity, preferable in a cell. Inhibiting microRNP activity refers to the inhibition of cleavage of the microRNA's target sequence or the repression of translation of the microRNA's target sequence. The method comprises introducing into the cell a single-stranded microRNA molecule.

Any anti-microRNA molecule can be used in the methods of the present invention, as long as the anti-microRNA is complementary, subject to the restrictions described above, to the microRNA present in the microRNP. Such anti-microRNAs include, for example, the anti-microRNA molecules mentioned above (see Table 1-4), and the anti-microRNAs molecules described in international PCT application number WO 03/029459 A2, the sequences of which are incorporated herein by reference.

The invention also includes any one of the microRNA molecules having the sequences as shown in Table 2. The novel microRNA molecules in Table 2 may optionally be modified as described above for anti-microRNA molecules. The other microRNA molecules in Tables 1, 3 and 4 are modified for increased nuclease resistance as described above for anti-microRNA molecules.

Utility

The anti-microRNA molecules and the microRNA molecules of the present invention have numerous in vivo, in vitro, and ex vivo applications.

For example, the anti-microRNA molecules and microRNA of the present invention may be used as a modulator of the expression of genes which are at least partially complementary to the anti-microRNA molecules and microRNA. For example, if a particular microRNA is beneficial for the survival of a cell, an appropriate isolated microRNA of the present invention may be introduced into the cell to promote survival. Alternatively, if a particular microRNA is harmful (e.g., induces apoptosis, induces cancer, etc.), an appropriate anti-microRNA molecule can be introduced into the cell in order to inhibit the activity of the microRNA and reduce the harm.

In addition, anti-microRNA molecules and/or microRNAs of-the present invention can be introduced into a cell to study the function of the microRNA. Any of the anti-microRNA molecules and/or microRNAs listed above can be introduced into a cell for studying their function. For example, a microRNA in a cell can be inhibited with a suitable anti-microRNA molecule. The function of the microRNA can be inferred by observing changes associated with inhibition of the microRNA in the cell in order to inhibit the activity of the microRNA and reduce the harm.

The cell can be any cell which expresses microRNA molecules, including the microRNA molecules listed herein. Alternatively, the cell can be any cell transfected with an expression vector containing the nucleotide sequence of a microRNA.

Examples of cells include, but are not limited to, endothelial cells, epithelial cells, leukocytes (e.g., T cells, B cells, neutrophils, macrophages, eosinophils, basophils, dendritic cells, natural killer cells and monocytes), stem cells, hemopoietic cells, embryonic cells, cancer cells.

The anti-microRNA molecules or microRNAs can be introduced into a cell by any method known to those skilled in the art. Useful delivery systems, include for example, liposomes and charged lipids. Liposomes typically encapsulate oligonucleotide molecules within their aqueous center. Charged lipids generally form lipid-oligonucleotide molecule complexes as a result of opposing charges.

These liposomes-oligonucleotide molecule complexes or lipid-oligonucleotide molecule complexes are usually internalized by endocytosis. The liposomes or charged lipids generally comprise helper lipids which disrupt the endosomal membrane and release the oligonucleotide molecules.

Other methods for introducing an anti-microRNA molecule or a microRNA into a cell include use of delivery vehicles, such as dendrimers, biodegradable polymers, polymers of amino acids, polymers of sugars, and oligonucleotide-binding nanoparticles. In addition, pluoronic gel as a depot reservoir can be used to deliver the anti-microRNA oligonucleotide molecules over a prolonged period. The above methods are described in, for example, Hughes et al., Drug Discovery Today 6, 303-315 (2001); Liang et al. Eur. J. Biochem. 269 5753-5758 (2002); and Becker et al., In Antisense Technology in the Central Nervous System (Leslie, R. A., Hunter, A. J. & Robertson, H. A., eds), pp. 147-157, Oxford University Press.

Targeting of an anti-microRNA molecule or a microRNA to a particular cell can be performed by any method known to those skilled in the art. For example, the anti-microRNA molecule or microRNA can be conjugated to an antibody or ligand specifically recognized by receptors on the cell.

The sequences of microRNA and anti-microRNA molecules are shown in Tables 1-4 below. Human sequences are indicated with the prefix “hsa.” Mouse sequences are indicated with the prefix “mmu.” Rat sequences are indicated with the prefix “rno.” C. elegan sequences are indicated with the prefix “cel.” Drosophila sequences are indicated with the prefix “dme.”

TABLE 1 Human, Mouse and Rat microRNA and anti-microRNA sequences. microRNA sequence Anti-microRNA molecule microRNA name (5′ to 3′) sequence (5′ to 3′) hsa-miR-100 SEQ ID NO. 1 SEQ ID NO. 307 AACCCGUAGAUCCGAACUUGUG CACAAGUUCGGAUCUACGGGUU hsa-miR-103 SEQ ID NO. 2 SEQ ID NO. 308 AGCAGCAUUGUACAGGGCUAUG CAUAGCCCUGUACAAUGCUGCU hsa-miR-105-5p SEQ ID NO. 3 SEQ ID NO. 309 UCAAAUGCUCAGACUCCUGUGG CCACAGGAGUCUGAGCAUUUGA hsa-miR-106a SEQ ID NO. 4 SEQ ID NO. 310 AAAAGUGCUUACAGUGCAGGUA UACCUGCACUGUAAGCACUUUU hsa-miR-106b SEQ ID NO. 5 SEQ ID NO. 311 UAAAGUGCUGACAGUGCAGAUA UAUCUGCACUGUCAGCACUUUA hsa-miR-107 SEQ ID NO. 6 SEQ ID NO. 312 AGCAGCAUUGUACAGGGCUAUC GAUAGCCCUGUACAAUGCUGCU hsa-miR-10b SEQ ID NO. 7 SEQ ID NO. 313 UACCCUGUAGAACCGAAUUUGU ACAAAUUCGGUUCUACAGGGUA hsa-miR-128b SEQ ID NO. 8 SEQ ID NO. 314 UCACAGUGAACCGGUCUCUUUC GAAAGAGACCGGUUCACUGUGA hsa-miR-130b SEQ ID NO. 9 SEQ ID NO. 315 CAGUGCAAUGAUGAAAGGGCAU AUGCCCUUUCAUCAUUGCACUG hsa-miR-140-3p SEQ ID NO. 10 SEQ ID NO. 316 UACCACAGGGUAGAACCACGGA UCCGUGGUUCUACCCUGUGGUA hsa-miR-142-5p SEQ ID NO. 11 SEQ ID NO. 317 CCCAUAAAGUAGAAAGCACUAC GUAGUGCUUUCUACUUUAUGGG hsa-miR-151-5p SEQ ID NO. 12 SEQ ID NO. 318 UCGAGGAGCUCACAGUCUAGUA UACUAGACUGUGAGCUCCUCGA hsa-miR-155 SEQ ID NO. 13 SEQ ID NO. 319 UUAAUGCUAAUCGUGAUAGGGG CCCCUAUCACGAUUAGCAUUAA hsa-miR-181a SEQ ID NO. 14 SEQ ID NO. 320 AACAUUCAACGCUGUCGGUGAG CUCACCGACAGCGUUGAAUGUU hsa-miR-181b SEQ ID NO. 15 SEQ ID NO. 321 AACAUUCAUUGCUGUCGGUGGG CCCACCGACAGCAAUGAAUGUU hsa-miR-181c SEQ ID NO. 16 SEQ ID NO. 322 AACAUUCAACCUGUCGGUGAGU ACUCACCGACAGGUUGAAUGUU hsa-miR-182 SEQ ID NO. 17 SEQ ID NO. 323 UUUGGCAAUGGUAGAACUCACA UGUGAGUUCUACCAUUGCCAAA hsa-miR-183 SEQ ID NO. 18 SEQ ID NO. 324 UAUGGCACUGGUAGAAUUCACU AGUGAAUUCUACCAGUGCCAUA hsa-miR-184 SEQ ID NO. 19 SEQ ID NO. 325 UGGACGGAGAACUGAUAAGGGU ACCCUUAUCAGUUCUCCGUCCA hsa-miR-185 SEQ ID NO. 20 SEQ ID NO. 326 UGGAGAGAAAGGCAGUUCCUGA UCAGGAACUGCCUUUCUCUCCA hsa-miR-186 SEQ ID NO. 21 SEQ ID NO. 327 CAAAGAAUUCUCCUUUUGGGCU AGCCCAAAAGGAGAAUUCUUUG hsa-miR-187 SEQ ID NO. 22 SEQ ID NO. 328 UCGUGUCUUGUGUUGCAGCCGG CCGGCUGCAACACAAGACACGA hsa-miR-188-3p SEQ ID NO. 23 SEQ ID NO. 329 CUCCCACAUGCAGGGUUUGCAG CUGCAAACCCUGCAUGUGGGAG hsa-miR-188-5p SEQ ID NO. 24 SEQ ID NO. 330 CAUCCCUUGCAUGGUGGAGGGU ACCCUCCACCAUGCAAGGGAUG hsa-miR-189 SEQ ID NO. 25 SEQ ID NO. 331 GUGCCUACUGAGCUGAUAUCAG CUGAUAUCAGCUCAGUAGGCAC hsa-miR-190 SEQ ID NO. 26 SEQ ID NO. 332 UGAUAUGUUUGAUAUAUUAGGU ACCUAAUAUAUCAAACAUAUCA hsa-miR-191 SEQ ID NO. 27 SEQ ID NO. 333 CAACGGAAUCCCAAAAGCAGCU AGCUGCUUUUGGGAUUCCGUUG hsa-miR-192 SEQ ID NO. 28 SEQ ID NO. 334 CUGACCUAUGAAUUGACAGCCA UGGCUGUCAAUUCAUAGGUCAG hsa-miR-193-3p SEQ ID NO. 29 SEQ ID NO. 335 AACUGGCCUACAAAGUCCCAGU ACUGGGACUUUGUAGGCCAGUU hsa-miR-193-5p SEQ ID NO. 30 SEQ ID NO. 336 UGGGUCUUUGCGGGCAAGAUGA UCAUCUUGCCCGCAAAGACCCA hsa-miR-194 SEQ ID NO. 31 SEQ ID NO. 337 UGUAACAGCAACUCCAUGUGGA UCCACAUGGAGUUGCUGUUACA hsa-miR-195 SEQ ID NO. 32 SEQ ID NO. 338 UAGCAGCACAGAAAUAUUGGCA UGCCAAUAUUUCUGUGCUGCUA hsa-miR-196 SEQ ID NO. 33 SEQ ID NO. 339 UAGGUAGUUUCAUGUUGUUGGG CCCAACAACAUGAAACUACCUA hsa-miR-197 SEQ ID NO. 34 SEQ ID NO. 340 UUCACCACCUUCUCCACCCAGC GCUGGGUGGAGAAGGUGGUGAA hsa-miR-198 SEQ ID NO. 35 SEQ ID NO. 341 GGUCCAGAGGGGAGAUAGGUUC GAACCUAUCUCCCCUCUGGACC hsa-miR-199a-3p SEQ ID NO. 36 SEQ ID NO. 342 ACAGUAGUCUGCACAUUGGUUA UAACCAAUGUGCAGACUACUGU hsa-miR-199a-5p SEQ ID NO. 37 SEQ ID NO. 343 CCCAGUGUUCAGACUACCUGUU AACAGGUAGUCUGAACACUGGG hsa-miR-199b SEQ ID NO. 38 SEQ ID NO. 344 CCCAGUGUUUAGACUAUCUGUU AACAGAUAGUCUAAACACUGGG hsa-miR-200a SEQ ID NO. 39 SEQ ID NO. 345 UAACACUGUCUGGUAACGAUGU ACAUCGUUACCAGACAGUGUUA hsa-miR-200b SEQ ID NO. 40 SEQ ID NO. 346 CUCUAAUACUGCCUGGUAAUGA UCAUUACCAGGCAGUAUUAGAG hsa-miR-200c SEQ ID NO. 41 SEQ ID NO. 347 AAUACUGCCGGGUAAUGAUGGA UCCAUCAUUACCCGGCAGUAUU hsa-miR-203 SEQ ID NO. 42 SEQ ID NO. 348 GUGAAAUGUUUAGGACCACUAG CUAGUGGUCCUAAACAUUUCAC hsa-miR-204 SEQ ID NO. 43 SEQ ID NO. 349 UUCCCUUUGUCAUCCUAUGCCU AGGCAUAGGAUGACAAAGGGAA hsa-miR-205 SEQ ID NO. 44 SEQ ID NO. 350 UCCUUCAUUCCACCGGAGUCUG CAGACUCCGGUGGAAUGAAGGA hsa-miR-206 SEQ ID NO. 45 SEQ ID NO. 351 UGGAAUGUAAGGAAGUGUGUGG CCACACACUUCCUUACAUUCCA hsa-miR-208 SEQ ID NO. 46 SEQ ID NO. 352 AUAAGACGAGCAAAAAGCUUGU ACAAGCUUUUUGCUCGUCUUAU hsa-miR-210 SEQ ID NO. 47 SEQ ID NO. 353 CUGUGCGUGUGACAGCGGCUGA UCAGCCGCUGUCACACGCACAG hsa-miR-211 SEQ ID NO. 48 SEQ ID NO. 354 UUCCCUUUGUCAUCCUUCGCCU AGGCGAAGGAUGACAAAGGGAA hsa-miR-212 SEQ ID NO. 49 SEQ ID NO. 355 UAACAGUCUCCAGUCACGGCCA UGGCCGUGACUGGAGACUGUUA hsa-miR-213 SEQ ID NO. 50 SEQ ID NO. 356 ACCAUCGACCGUUGAUUGUACC GGUACAAUCAACGGUCGAUGGU hsa-miR-214 SEQ ID NO. 51 SEQ ID NO. 357 ACAGCAGGCACAGACAGGCAGU ACUGCCUGUCUGUGCCUGCUGU hsa-miR-215 SEQ ID NO. 52 SEQ ID NO. 358 AUGACCUAUGAAUUGACAGACA UGUCUGUCAAUUCAUAGGUCAU hsa-miR-216 SEQ ID NO. 53 SEQ ID NO. 359 UAAUCUCAGCUGGCAACUGUGA UCACAGUUGCCAGCUGAGAUUA hsa-miR-217 SEQ ID NO. 54 SEQ ID NO. 360 UACUGCAUCAGGAACUGAUUGG CCAAUCAGUUCCUGAUGCAGUA hsa-miR-218 SEQ ID NO. 55 SEQ ID NO. 361 UUGUGCUUGAUCUAACCAUGUG CACAUGGUUAGAUCAAGCACAA hsa-miR-219 SEQ ID NO. 56 SEQ ID NO. 362 UGAUUGUCCAAACGCAAUUCUU AAGAAUUGCGUUUGGACAAUCA hsa-miR-220 SEQ ID NO. 57 SEQ ID NO. 363 CCACACCGUAUCUGACACUUUG CAAAGUGUCAGAUACGGUGUGG hsa-miR-221 SEQ ID NO. 58 SEQ ID NO. 364 AGCUACAUUGUCUGCUGGGUUU AAACCCAGCAGACAAUGUAGCU hsa-miR-222 SEQ ID NO. 59 SEQ ID NO. 365 AGCUACAUCUGGCUACUGGGUC GACCCAGUAGCCAGAUGUAGCU hsa-miR-223 SEQ ID NO. 60 SEQ ID NO. 366 UGUCAGUUUGUCAAAUACCCCA UGGGGUAUUUGACAAACUGACA hsa-miR-224 SEQ ID NO. 61 SEQ ID NO. 367 CAAGUCACUAGUGGUUCCGUUU AAACGGAACCACUAGUGACUUG hsa-miR-28-5p SEQ ID NO. 62 SEQ ID NO. 368 AAGGAGCUCACAGUCUAUUGAG CUCAAUAGACUGUGAGCUCCUU hsa-miR-290 SEQ ID NO. 63 SEQ ID NO. 369 CUCAAACUGUGGGGGCACUUUC GAAAGUGCCCCCACAGUUUGAG hsa-miR-296 SEQ ID NO. 64 SEQ ID NO. 370 AGGGCCCCCCCUCAAUCCUGUU AACAGGAUUGAGGGGGGGCCCU hsa-miR-299 SEQ ID NO. 65 SEQ ID NO. 371 UGGUUUACCGUCCCACAUACAU AUGUAUGUGGGACGGUAAACCA hsa-miR-301 SEQ ID NO. 66 SEQ ID NO. 372 CAGUGCAAUAGUAUUGUCAAAG CUUUGACAAUACUAUUGCACUG hsa-miR-302 SEQ ID NO. 67 SEQ ID NO. 373 UAAGUGCUUCCAUGUUUUGGUG CACCAAAACAUGGAAGCACUUA hsa-miR-30e SEQ ID NO. 68 SEQ ID NO. 374 UGUAAACAUCCUUGACUGGAAG CUUCCAGUCAAGGAUGUUUACA hsa-miR-320 SEQ ID NO. 69 SEQ ID NO. 375 AAAAGCUGGGUUGAGAGGGCGA UCGCCCUCUCAACCCAGCUUUU hsa-miR-321 SEQ ID NO. 70 SEQ ID NO. 376 UAAGCCAGGGAUUGUGGGUUCG CGAACCCACAAUCCCUGGCUUA hsa-miR-322 SEQ ID NO. 71 SEQ ID NO. 377 AAACAUGAAUUGCUGCUGUAUC GAUACAGCAGCAAUUCAUGUUU hsa-miR-323 SEQ ID NO. 72 SEQ ID NO. 378 GCACAUUACACGGUCGACCUCU AGAGGUCGACCGUGUAAUGUGC hsa-miR-324-3p SEQ ID NO. 73 SEQ ID NO. 379 CCACUGCCCCAGGUGCUGCUGG CCAGCAGCACCUGGGGCAGUGG hsa-miR-324-5p SEQ ID NO. 74 SEQ ID NO. 380 CGCAUCCCCUAGGGCAUUGGUG CACCAAUGCCCUAGGGGAUGCG hsa-miR-326 SEQ ID NO. 75 SEQ ID NO. 381 CCUCUGGGCCCUUCCUCCAGCC GGCUGGAGGAAGGGCCCAGAGG hsa-miR-328 SEQ ID NO. 76 SEQ ID NO. 382 CUGGCCCUCUCUGCCCUUCCGU ACGGAAGGGCAGAGAGGGCCAG hsa-miR-329 SEQ ID NO. 77 SEQ ID NO. 383 AACACACCCAGCUAACCUUUUU AAAAAGGUUAGCUGGGUGUGUU hsa-miR-34a SEQ ID NO. 78 SEQ ID NO. 384 UGGCAGUGUCUUAGCUGGUUGU ACAACCAGCUAAGACACUGCCA hsa-miR-34b SEQ ID NO. 79 SEQ ID NO. 385 AGGCAGUGUCAUUAGCUGAUUG CAAUCAGCUAAUGACACUGCCU hsa-miR-34c SEQ ID NO. 80 SEQ ID NO. 386 AGGCAGUGUAGUUAGCUGAUUG CAAUCAGCUAACUACACUGCCU hsa-miR-92 SEQ ID NO. 81 SEQ ID NO. 387 UAUUGCACUUGUCCCGGCCUGU ACAGGCCGGGACAAGUGCAAUA hsa-miR-93 SEQ ID NO. 82 SEQ ID NO. 388 AAAGUGCUGUUCGUGCAGGUAG CUACCUGCACGAACAGCACUUU hsa-miR-95 SEQ ID NO. 83 SEQ ID NO. 389 UUCAACGGGUAUUUAUUGAGCA UGCUCAAUAAAUACCCGUUGAA hsa-miR-96 SEQ ID NO. 84 SEQ ID NO. 390 UUUGGCACUAGCACAUUUUUGC GCAAAAAUGUGCUAGUGCCAAA hsa-miR-98 SEQ ID NO. 85 SEQ ID NO. 391 UGAGGUAGUAAGUUGUAUUGUU AACAAUACAACUUACUACCUCA mmu-miR-106a SEQ ID NO. 86 SEQ ID NO. 392 CAAAGUGCUAACAGUGCAGGUA UACCUGCACUGUUAGCACUUUG mmu-miR-10b SEQ ID NO. 87 SEQ ID NO. 393 CCCUGUAGAACCGAAUUUGUGU ACACAAAUUCGGUUCUACAGGG mmu-miR-135b SEQ ID NO. 88 SEQ ID NO. 394 UAUGGCUUUUCAUUCCUAUGUG CACAUAGGAAUGAAAAGCCAUA mmu-miR-148b SEQ ID NO. 89 SEQ ID NO. 395 UCAGUGCAUCACAGAACUUUGU ACAAAGUUCUGUGAUGCACUGA mmu-miR-151-3p SEQ ID NO. 90 SEQ ID NO. 396 CUAGACUGAGGCUCCUUGAGGA UCCUCAAGGAGCCUCAGUCUAG mmu-miR-155 SEQ ID NO. 91 SEQ ID NO. 397 UUAAUGCUAAUUGUGAUAGGGG CCCCUAUCACAAUUAGCAUUAA mmu-miR-199b SEQ ID NO. 92 SEQ ID NO. 398 CCCAGUGUUUAGACUACCUGUU AACAGGUAGUCUAAACACUGGG mmu-miR-200b SEQ ID NO. 93 SEQ ID NO. 399 UAAUACUGCCUGGUAAUGAUGA UCAUCAUUACCAGGCAGUAUUA mmu-miR-203 SEQ ID NO. 94 SEQ ID NO. 400 UGAAAUGUUUAGGACCACUAGA UCUAGUGGUCCUAAACAUUUCA mmu-miR-211 SEQ ID NO. 95 SEQ ID NO. 401 UUCCCUUUGUCAUCCUUUGCCU AGGCAAAGGAUGACAAAGGGAA mmu-miR-217 SEQ ID NO. 96 SEQ ID NO. 402 UACUGCAUCAGGAACUGACUGG CCAGUCAGUUCCUGAUGCAGUA mmu-miR-224 SEQ ID NO. 97 SEQ ID NO. 403 UAAGUCACUAGUGGUUCCGUUU AAACGGAACCACUAGUGACUUA mmu-miR-28-3p SEQ ID NO. 98 SEQ ID NO. 404 CACUAGAUUGUGAGCUGCUGGA UCCAGCAGCUCACAAUCUAGUG mmu-miR-290 SEQ ID NO. 99 SEQ ID NO. 405 CUCAAACUAUGGGGGCACUUUU AAAAGUGCCCCCAUAGUUUGAG mmu-miR-291-3p SEQ ID NO. 100 SEQ ID NO. 406 AAAGUGCUUCCACUUUGUGUGC GCACACAAAGUGGAAGCACUUU mmu-miR-291-5p SEQ ID NO. 101 SEQ ID NO. 407 CAUCAAAGUGGAGGCCCUCUCU AGAGAGGGCCUCCACUUUGAUG mmu-miR-292-3p SEQ ID NO. 102 SEQ ID NO. 408 AAGUGCCGCCAGGUUUUGAGUG CACUCAAAACCUGGCGGCACUU mmu-miR-292-5p SEQ ID NO. 103 SEQ ID NO. 409 ACUCAAACUGGGGGCUCUUUUG CAAAAGAGCCCCCAGUUUGAGU mmu-miR-293 SEQ ID NO. 104 SEQ ID NO. 410 AGUGCCGCAGAGUUUGUAGUGU ACACUACAAACUCUGCGGCACU mmu-miR-294 SEQ ID NO. 105 SEQ ID NO. 411 AAAGUGCUUCCCUUUUGUGUGU ACACACAAAAGGGAAGCACUUU mmu-miR-295 SEQ ID NO. 106 SEQ ID NO. 412 AAAGUGCUACUACUUUUGAGUC GACUCAAAAGUAGUAGCACUUU mmu-miR-297 SEQ ID NO. 107 SEQ ID NO. 413 AUGUAUGUGUGCAUGUGCAUGU ACAUGCACAUGCACACAUACAU mmu-miR-298 SEQ ID NO. 108 SEQ ID NO. 414 GGCAGAGGAGGGCUGUUCUUCC GGAAGAACAGCCCUCCUCUGCC mmu-miR-300 SEQ ID NO. 109 SEQ ID NO. 415 UAUGCAAGGGCAAGCUCUCUUC GAAGAGAGCUUGCCCUUGCAUA mmu-miR-31 SEQ ID NO. 110 SEQ ID NO. 416 AGGCAAGAUGCUGGCAUAGCUG CAGCUAUGCCAGCAUCUUGCCU mmu-miR-322 SEQ ID NO. 111 SEQ ID NO. 417 AAACAUGAAGCGCUGCAACACC GGUGUUGCAGCGCUUCAUGUUU mmu-miR-325 SEQ ID NO. 112 SEQ ID NO. 418 CCUAGUAGGUGCUCAGUAAGUG CACUUACUGAGCACCUACUAGG mmu-miR-326 SEQ ID NO. 113 SEQ ID NO. 419 CCUCUGGGCCCUUCCUCCAGUC GACUGGAGGAAGGGCCCAGAGG mmu-miR-330 SEQ ID NO. 114 SEQ ID NO. 420 GCAAAGCACAGGGCCUGCAGAG CUCUGCAGGCCCUGUGCUUUGC mmu-miR-331 SEQ ID NO. 115 SEQ ID NO. 421 GCCCCUGGGCCUAUCCUAGAAC GUUCUAGGAUAGGCCCAGGGGC mmu-miR-337 SEQ ID NO. 116 SEQ ID NO. 422 UUCAGCUCCUAUAUGAUGCCUU AAGGCAUCAUAUAGGAGCUGAA mmu-miR-338 SEQ ID NO. 117 SEQ ID NO. 423 UCCAGCAUCAGUGAUUUUGUUG CAACAAAAUCACUGAUGCUGGA mmu-miR-339 SEQ ID NO. 118 SEQ ID NO. 424 UCCCUGUCCUCCAGGAGCUCAC GUGAGCUCCUGGAGGACAGGGA mmu-miR-340 SEQ ID NO. 119 SEQ ID NO. 425 UCCGUCUCAGUUACUUUAUAGC GCUAUAAAGUAACUGAGACGGA mmu-miR-341 SEQ ID NO. 120 SEQ ID NO. 426 UCGAUCGGUCGGUCGGUCAGUC GACUGACCGACCGACCGAUCGA mmu-miR-342 SEQ ID NO. 121 SEQ ID NO. 427 UCUCACACAGAAAUCGCACCCG CGGGUGCGAUUUCUGUGUGAGA mmu-miR-344 SEQ ID NO. 122 SEQ ID NO. 428 UGAUCUAGCCAAAGCCUGACUG CAGUCAGGCUUUGGCUAGAUCA mmu-miR-345 SEQ ID NO. 123 SEQ ID NO. 429 UGCUGACCCCUAGUCCAGUGCU AGCACUGGACUAGGGGUCAGCA mmu-miR-346 SEQ ID NO. 124 SEQ ID NO. 430 UGUCUGCCCGAGUGCCUGCCUC GAGGCAGGCACUCGGGCAGACA mmu-miR-34b SEQ ID NO. 125 SEQ ID NO. 431 UAGGCAGUGUAAUUAGCUGAUU AAUCAGCUAAUUACACUGCCUA mmu-miR-350 SEQ ID NO. 126 SEQ ID NO. 432 UUCACAAAGCCCAUACACUUUC GAAAGUGUAUGGGCUUUGUGAA mmu-miR-351 SEQ ID NO. 127 SEQ ID NO. 433 UCCCUGAGGAGCCCUUUGAGCC GGCUCAAAGGGCUCCUCAGGGA mmu-miR-7b SEQ ID NO. 128 SEQ ID NO. 434 UGGAAGACUUGUGAUUUUGUUG CAACAAAAUCACAAGUCUUCCA mmu-miR-92 SEQ ID NO. 129 SEQ ID NO. 435 UAUUGCACUUGUCCCGGCCUGA UCAGGCCGGGACAAGUGCAAUA mmu-miR-93 SEQ ID NO. 130 SEQ ID NO. 436 CAAAGUGCUGUUCGUGCAGGUA UACCUGCACGAACAGCACUUUG rno-miR-327 SEQ ID NO. 131 SEQ ID NO. 437 CCUUGAGGGGCAUGAGGGUAGU ACUACCCUCAUGCCCCUCAAGG rno-miR-333 SEQ ID NO. 132 SEQ ID NO. 438 GUGGUGUGCUAGUUACUUUUGG CCAAAAGUAACUAGCACACCAC rno-miR-335 SEQ ID NO. 133 SEQ ID NO. 439 UCAAGAGCAAUAACGAAAAAUG CAUUUUUCGUUAUUGCUCUUGA rno-miR-336 SEQ ID NO. 134 SEQ ID NO. 440 UCACCCUUCCAUAUCUAGUCUC GAGACUAGAUAUGGAAGGGUGA rno-miR-343 SEQ ID NO. 135 SEQ ID NO. 441 UCUCCCUCCGUGUGCCCAGUAU AUACUGGGCACACGGAGGGAGA rno-miR-347 SEQ ID NO. 136 SEQ ID NO. 442 UGUCCCUCUGGGUCGCCCAGCU AGCUGGGCGACCCAGAGGGACA rno-miR-349 SEQ ID NO. 137 SEQ ID NO. 443 CAGCCCUGCUGUCUUAACCUCU AGAGGUUAAGACAGCAGGGCUG rno-miR-352 SEQ ID NO. 138 SEQ ID NO. 444 AGAGUAGUAGGUUGCAUAGUAC GUACUAUGCAACCUACUACUCU

TABLE 2 Novel Human microRNA and anti-microRNA sequences. microRNA sequence Anti-microRNA molecule microRNA name (5′ to 3′) sequence (5′ to 3′) hsa-miR-361 SEQ ID NO. 139 SEQ ID NO. 445 UUAUCAGAAUCUCCAGGGGUAC GUACCCCUGGAGAUUCUGAUAA hsa-miR-362 SEQ ID NO. 140 SEQ ID NO. 446 AAUCCUUGGAACCUAGGUGUGA UCACACCUAGGUUCCAAGGAUU hsa-miR-363 SEQ ID NO. 141 SEQ ID NO. 447 AUUGCACGGUAUCCAUCUGUAA UUACAGAUGGAUACCGUGCAAU hsa-miR-364 SEQ ID NO. 142 SEQ ID NO. 448 CGGCGGGGACGGCGAUUGGUCC GGACCAAUCGCCGUCCCCGCCG hsa-miR-365 SEQ ID NO. 143 SEQ ID NO. 449 UAAUGCCCCUAAAAAUCCUUAU AUAAGGAUUUUUAGGGGCAUUA hsa-miR-366 SEQ ID NO. 144 SEQ ID NO. 450 UAACUGGUUGAACAACUGAACC GGUUCAGUUGUUCAACCAGUUA

TABLE 3 C. elegans microRNA and anti-microRNA sequences. microRNA sequence Anti-microRNA molecule microRNA name (5′ to 3′) sequence (5′ to 3′) Cel-let-7 SEQ ID NO. 145 SEQ ID NO. 451 UGAGGUAGUAGGUUGUAUAGUU AACUAUACAACCUACUACCUCA CeL-lin-4 SEQ ID NO. 146 SEQ ID NO. 452 UCCCUGAGACCUCAAGUGUGAG CUCACACUUGAGGUCUCAGGGA Cel-miR-1 SEQ ID NO. 147 SEQ ID NO. 453 UGGAAUGUAAAGAAGUAUGUAG CUACAUACUUCUUUACAUUCCA Cel-miR-2 SEQ ID NO. 148 SEQ ID NO. 454 UAUCACAGCCAGCUUUGAUGUG CACAUCAAAGCUGGCUGUGAUA Cel-miR-34 SEQ ID NO. 149 SEQ ID NO. 455 AGGCAGUGUGGUUAGCUGGUUG CAACCAGCUAACCACACUGCCU Cel-miR-35 SEQ ID NO. 150 SEQ ID NO. 456 UCACCGGGUGGAAACUAGCAGU ACUGCUAGUUUCCACCCGGUGA Cel-miR-36 SEQ ID NO. 151 SEQ ID NO. 457 UCACCGGGUGAAAAUUCGCAUG CAUGCGAAUUUUCACCCGGUGA Cel-miR-37 SEQ ID NO. 152 SEQ ID NO. 458 UCACCGGGUGAACACUUGCAGU ACUGCAAGUGUUCACCCGGUGA Cel-miR-38 SEQ ID NO. 153 SEQ ID NO. 459 UCACCGGGAGAAAAACUGGAGU ACUCCAGUUUUUCUCCCGGUGA Cel-miR-39 SEQ ID NO. 154 SEQ ID NO. 460 UCACCGGGUGUAAAUCAGCUUG CAAGCUGAUUUACACCCGGUGA Cel-miR-40 SEQ ID NO. 155 SEQ ID NO. 461 UCACCGGGUGUACAUCAGCUAA UUAGCUGAUGUACACCCGGUGA Cel-miR-41 SEQ ID NO. 156 SEQ ID NO. 462 UCACCGGGUGAAAAAUCACCUA UAGGUGAUUUUUCACCCGGUGA Cel-miR-42 SEQ ID NO. 157 SEQ ID NO. 463 CACCGGGUUAACAUCUACAGAG CUCUGUAGAUGUUAACCCGGUG Cel-miR-43 SEQ ID NO. 158 SEQ ID NO. 464 UAUCACAGUUUACUUGCUGUCG CGACAGCAAGUAAACUGUGAUA Cel-miR-44 SEQ ID NO. 159 SEQ ID NO. 465 UGACUAGAGACACAUUCAGCUU AAGCUGAAUGUGUCUCUAGUCA Cel-miR-45 SEQ ID NO. 160 SEQ ID NO. 466 UGACUAGAGACACAUUCAGCUU AAGCUGAAUGUGUCUCUAGUCA Cel-miR-46 SEQ ID NO. 161 SEQ ID NO. 467 UGUCAUGGAGUCGCUCUCUUCA UGAAGAGAGCGACUCCAUGACA Cel-miR-47 SEQ ID NO. 162 SEQ ID NO. 468 UGUCAUGGAGGCGCUCUCUUCA UGAAGAGAGCGCCUCCAUGACA Cel-miR-48 SEQ ID NO. 163 SEQ ID NO. 469 UGAGGUAGGCUCAGUAGAUGCG CGCAUCUACUGAGCCUACCUCA Cel-miR-49 SEQ ID NO. 164 SEQ ID NO. 470 AAGCACCACGAGAAGCUGCAGA UCUGCAGCUUCUCGUGGUGCUU Cel-miR-50 SEQ ID NO. 165 SEQ ID NO. 471 UGAUAUGUCUGGUAUUCUUGGG CCCAAGAAUACCAGACAUAUCA Cel-miR-51 SEQ ID NO. 166 SEQ ID NO. 472 UACCCGUAGCUCCUAUCCAUGU ACAUGGAUAGGAGCUACGGGUA Cel-miR-52 SEQ ID NO. 167 SEQ ID NO. 473 CACCCGUACAUAUGUUUCCGUG CACGGAAACAUAUGUACGGGUG Cel-miR-53 SEQ ID NO. 168 SEQ ID NO. 474 CACCCGUACAUUUGUUUCCGUG CACGGAAACAAAUGUACGGGUG Cel-miR-54 SEQ ID NO. 169 SEQ ID NO. 475 UACCCGUAAUCUUCAUAAUCCG CGGAUUAUGAAGAUUACGGGUA Cel-miR-55 SEQ ID NO. 170 SEQ ID NO. 476 UACCCGUAUAAGUUUCUGCUGA UCAGCAGAAACUUAUACGGGUA Cel-miR-56 SEQ ID NO. 171 SEQ ID NO. 477 UACCCGUAAUGUUUCCGCUGAG CUCAGCGGAAACAUUACGGGUA Cel-miR-57 SEQ ID NO. 172 SEQ ID NO. 478 UACCCUGUAGAUCGAGCUGUGU ACACAGCUCGAUCUACAGGGUA Cel-miR-58 SEQ ID NO. 173 SEQ ID NO. 479 UGAGAUCGUUCAGUACGGCAAU AUUGCCGUACUGAACGAUCUCA Cel-miR-59 SEQ ID NO. 174 SEQ ID NO. 480 UCGAAUCGUUUAUCAGGAUGAU AUCAUCCUGAUAAACGAUUCGA Cel-miR-60 SEQ ID NO. 175 SEQ ID NO. 481 UAUUAUGCACAUUUUCUAGUUC GAACUAGAAAAUGUGCAUAAUA Cel-miR-61 SEQ ID NO. 176 SEQ ID NO. 482 UGACUAGAACCGUUACUCAUCU AGAUGAGUAACGGUUCUAGUCA Cel-miR-62 SEQ ID NO. 177 SEQ ID NO. 483 UGAUAUGUAAUCUAGCUUACAG CUGUAAGCUAGAUUACAUAUCA Cel-miR-63 SEQ ID NO. 178 SEQ ID NO. 484 AUGACACUGAAGCGAGUUGGAA UUCCAACUCGCUUCAGUGUCAU Cel-miR-64 SEQ ID NO. 179 SEQ ID NO. 485 UAUGACACUGAAGCGUUACCGA UCGGUAACGCUUCAGUGUCAUA Cel-miR-65 SEQ ID NO. 180 SEQ ID NO. 486 UAUGACACUGAAGCGUAACCGA UCGGUUACGCUUCAGUGUCAUA Cel-miR-66 SEQ ID NO. 181 SEQ ID NO. 487 CAUGACACUGAUUAGGGAUGUG CACAUCCCUAAUCAGUGUCAUG Cel-miR-67 SEQ ID NO. 182 SEQ ID NO. 488 UCACAACCUCCUAGAAAGAGUA UACUCUUUCUAGGAGGUUGUGA Cel-miR-68 SEQ ID NO. 183 SEQ ID NO. 489 UCGAAGACUCAAAAGUGUAGAC GUCUACACUUUUGAGUCUUCGA Cel-miR-69 SEQ ID NO. 184 SEQ ID NO. 490 UCGAAAAUUAAAAAGUGUAGAA UUCUACACUUUUUAAUUUUCGA Cel-miR-70 SEQ ID NO. 185 SEQ ID NO. 491 UAAUACGUCGUUGGUGUUUCCA UGGAAACACCAACGACGUAUUA Cel-miR-71 SEQ ID NO. 186 SEQ ID NO. 492 UGAAAGACAUGGGUAGUGAACG CGUUCACUACCCAUGUCUUUCA Cel-miR-72 SEQ ID NO. 187 SEQ ID NO. 493 AGGCAAGAUGUUGGCAUAGCUG CAGCUAUGCCAACAUCUUGCCU Cel-miR-73 SEQ ID NO. 188 SEQ ID NO. 494 UGGCAAGAUGUAGGCAGUUCAG CUGAACUGCCUACAUCUUGCCA Cel-miR-74 SEQ ID NO. 189 SEQ ID NO. 495 UGGCAAGAAAUGGCAGUCUACA UGUAGACUGCCAUUUCUUGCCA Cel-miR-75 SEQ ID NO. 190 SEQ ID NO. 496 UUAAAGCUACCAACCGGCUUCA UGAAGCCGGUUGGUAGCUUUAA Cel-miR-76 SEQ ID NO. 191 SEQ ID NO. 497 UUCGUUGUUGAUGAAGCCUUGA UCAAGGCUUCAUCAACAACGAA Cel-miR-77 SEQ ID NO. 192 SEQ ID NO. 498 UUCAUCAGGCCAUAGCUGUCCA UGGACAGCUAUGGCCUGAUGAA Cel-miR-78 SEQ ID NO. 193 SEQ ID NO. 499 UGGAGGCCUGGUUGUUUGUGCU AGCACAAACAACCAGGCCUCCA Cel-miR-79 SEQ ID NO. 194 SEQ ID NO. 500 AUAAAGCUAGGUUACCAAAGCU AGCUUUGGUAACCUAGCUUUAU Cel-miR-227 SEQ ID NO. 195 SEQ ID NO. 501 AGCUUUCGACAUGAUUCUGAAC GUUCAGAAUCAUGUCGAAAGCU Cel-miR-80 SEQ ID NO. 196 SEQ ID NO. 502 UGAGAUCAUUAGUUGAAAGCCG CGGCUUUCAACUAAUGAUCUCA Cel-miR-81 SEQ ID NO. 197 SEQ ID NO. 503 UGAGAUCAUCGUGAAAGCUAGU ACUAGCUUUCACGAUGAUCUCA Cel-miR-82 SEQ ID NO. 198 SEQ ID NO. 504 UGAGAUCAUCGUGAAAGCCAGU ACUGGCUUUCACGAUGAUCUCA Cel-miR-83 SEQ ID NO. 199 SEQ ID NO. 505 UAGCACCAUAUAAAUUCAGUAA UUACUGAAUUUAUAUGGUGCUA Cel-miR-84 SEQ ID NO. 200 SEQ ID NO. 506 UGAGGUAGUAUGUAAUAUUGUA UACAAUAUUACAUACUACCUCA Cel-miR-85 SEQ ID NO. 201 SEQ ID NO. 507 UACAAAGUAUUUGAAAAGUCGU ACGACUUUUCAAAUACUUUGUA Cel-miR-86 SEQ ID NO. 202 SEQ ID NO. 508 UAAGUGAAUGCUUUGCCACAGU ACUGUGGCAAAGCAUUCACUUA Cel-miR-87 SEQ ID NO. 203 SEQ ID NO. 509 GUGAGCAAAGUUUCAGGUGUGC GCACACCUGAAACUUUGCUCAC Cel-miR-90 SEQ ID NO. 204 SEQ ID NO. 510 UGAUAUGUUGUUUGAAUGCCCC GGGGCAUUCAAACAACAUAUCA Cel-miR-124 SEQ ID NO. 205 SEQ ID NO. 511 UAAGGCACGCGGUGAAUGCCAC GUGGCAUUCACCGCGUGCCUUA Cel-miR-228 SEQ ID NO. 206 SEQ ID NO. 512 AAUGGCACUGCAUGAAUUCACG CGUGAAUUCAUGCAGUGCCAUU Cel-miR-229 SEQ ID NO. 207 SEQ ID NO. 513 AAUGACACUGGUUAUCUUUUCC GGAAAAGAUAACCAGUGUCAUU Cel-miR-230 SEQ ID NO. 208 SEQ ID NO. 514 GUAUUAGUUGUGCGACCAGGAG CUCCUGGUCGCACAACUAAUAC Cel-miR-231 SEQ ID NO. 209 SEQ ID NO. 515 UAAGCUCGUGAUCAACAGGCAG CUGCCUGUUGAUCACGAGCUUA Cel-miR-232 SEQ ID NO. 210 SEQ ID NO. 516 UAAAUGCAUCUUAACUGCGGUG CACCGCAGUUAAGAUGCAUUUA Cel-miR-233 SEQ ID NO. 211 SEQ ID NO. 517 UUGAGCAAUGCGCAUGUGCGGG CCCGCACAUGCGCAUUGCUCAA Cel-miR-234 SEQ ID NO. 212 SEQ ID NO. 518 UUAUUGCUCGAGAAUACCCUUU AAAGGGUAUUCUCGAGCAAUAA Cel-miR-235 SEQ ID NO. 213 SEQ ID NO. 519 UAUUGCACUCUCCCCGGCCUGA UCAGGCCGGGGAGAGUGCAAUA Cel-miR-236 SEQ ID NO. 214 SEQ ID NO. 520 UAAUACUGUCAGGUAAUGACGC GCGUCAUUACCUGACAGUAUUA Cel-miR-237 SEQ ID NO. 215 SEQ ID NO. 521 UCCCUGAGAAUUCUCGAACAGC GCUGUUCGAGAAUUCUCAGGGA Cel-miR-238 SEQ ID NO. 216 SEQ ID NO. 522 UUUGUACUCCGAUGCCAUUCAG CUGAAUGGCAUCGGAGUACAAA Cel-miR-239a SEQ ID NO. 217 SEQ ID NO. 523 UUUGUACUACACAUAGGUACUG CAGUACCUAUGUGUAGUACAAA Cel-miR-239b SEQ ID NO. 218 SEQ ID NO. 524 UUUGUACUACACAAAAGUACUG CAGUACUUUUGUGUAGUACAAA Cel-miR-240 SEQ ID NO. 219 SEQ ID NO. 525 UACUGGCCCCCAAAUCUUCGCU AGCGAAGAUUUGGGGGCCAGUA Cel-miR-241 SEQ ID NO. 220 SEQ ID NO. 526 UGAGGUAGGUGCGAGAAAUGAC GUCAUUUCUCGCACCUACCUCA Cel-miR-242 SEQ ID NO. 221 SEQ ID NO. 527 UUGCGUAGGCCUUUGCUUCGAG CUCGAAGCAAAGGCCUACGCAA Cel-miR-243 SEQ ID NO. 222 SEQ ID NO. 528 CGGUACGAUCGCGGCGGGAUAU AUAUCCCGCCGCGAUCGUACCG Cel-miR-244 SEQ ID NO. 223 SEQ ID NO. 529 UCUUUGGUUGUACAAAGUGGUA UACCACUUUGUACAACCAAAGA Cel-miR-245 SEQ ID NO. 224 SEQ ID NO. 530 AUUGGUCCCCUCCAAGUAGCUC GAGCUACUUGGAGGGGACCAAU Cel-miR-246 SEQ ID NO. 225 SEQ ID NO. 531 UUACAUGUUUCGGGUAGGAGCU AGCUCCUACCCGAAACAUGUAA Cel-miR-247 SEQ ID NO. 226 SEQ ID NO. 532 UGACUAGAGCCUAUUCUCUUCU AGAAGAGAAUAGGCUCUAGUCA Cel-miR-248 SEQ ID NO. 227 SEQ ID NO. 533 UACACGUGCACGGAUAACGCUC GAGCGUUAUCCGUGCACGUGUA Cel-miR-249 SEQ ID NO. 228 SEQ ID NO. 534 UCACAGGACUUUUGAGCGUUGC GCAACGCUCAAAAGUCCUGUGA Cel-miR-250 SEQ ID NO. 229 SEQ ID NO. 535 UCACAGUCAACUGUUGGCAUGG CCAUGCCAACAGUUGACUGUGA Cel-miR-251 SEQ ID NO. 230 SEQ ID NO. 536 UUAAGUAGUGGUGCCGCUCUUA UAAGAGCGGCACCACUACUUAA Cel-miR-252 SEQ ID NO. 231 SEQ ID NO. 537 UAAGUAGUAGUGCCGCAGGUAA UUACCUGCGGCACUACUACUUA Cel-miR-253 SEQ ID NO. 232 SEQ ID NO. 538 CACACCUCACUAACACUGACCA UGGUCAGUGUUAGUGAGGUGUG Cel-miR-254 SEQ ID NO. 233 SEQ ID NO. 539 UGCAAAUCUUUCGCGACUGUAG CUACAGUCGCGAAAGAUUUGCA Cel-miR-256 SEQ ID NO. 234 SEQ ID NO. 540 UGGAAUGCAUAGAAGACUGUAC GUACAGUCUUCUAUGCAUUCCA Cel-miR-257 SEQ ID NO. 235 SEQ ID NO. 541 GAGUAUCAGGAGUACCCAGUGA UCACUGGGUACUCCUGAUACUC Cel-miR-258 SEQ ID NO. 236 SEQ ID NO. 542 GGUUUUGAGAGGAAUCCUUUUA UAAAAGGAUUCCUCUCAAAACC Cel-miR-259 SEQ ID NO. 237 SEQ ID NO. 543 AGUAAAUCUCAUCCUAAUCUGG CCAGAUUAGGAUGAGAUUUACU Cel-miR-260 SEQ ID NO. 238 SEQ ID NO. 544 GUGAUGUCGAACUCUUGUAGGA UCCUACAAGAGUUCGACAUCAC Cel-miR-261 SEQ ID NO. 239 SEQ ID NO. 545 UAGCUUUUUAGUUUUCACGGUG CACCGUGAAAACUAAAAGCUA Cel-miR-262 SEQ ID NO. 240 SEQ ID NO. 546 GUUUCUCGAUGUUUUCUGAUAC GUAUCAGAAAACAUCGAGAAAC Cel-miR-264 SEQ ID NO. 241 SEQ ID NO. 547 GGCGGGUGGUUGUUGUUAUGGG CCCAUAACAACAACCACCCGCC Cel-miR-265 SEQ ID NO. 242 SEQ ID NO. 548 UGAGGGAGGAAGGGUGGUAUUU AAAUACCACCCUUCCUCCCUCA Cel-miR-266 SEQ ID NO. 243 SEQ ID NO. 549 AGGCAAGACUUUGGCAAAGCUU AAGCUUUGCCAAAGUCUUGCCU Cel-miR-267 SEQ ID NO. 244 SEQ ID NO. 550 CCCGUGAAGUGUCUGCUGCAAU AUUGCAGCAGACACUUCACGGG Cel-miR-268 SEQ ID NO. 245 SEQ ID NO. 551 GGCAAGAAUUAGAAGCAGUUUG CAAACUGCUUCUAAUUCUUGCC Cel-miR-269 SEQ ID NO. 246 SEQ ID NO. 552 GGCAAGACUCUGGCAAAACUUG CAAGUUUUGCCAGAGUCUUGCC Cel-miR-270 SEQ ID NO. 247 SEQ ID NO. 553 GGCAUGAUGUAGCAGUGGAGAU AUCUCCACUGCUACAUCAUGCC Cel-miR-271 SEQ ID NO. 248 SEQ ID NO. 554 UCGCCGGGUGGGAAAGCAUUCG CGAAUGCUUUCCCACCCGGCGA Cel-miR-272 SEQ ID NO. 249 SEQ ID NO. 555 UGUAGGCAUGGGUGUUUGGAAG CUUCCAAACACCCAUGCCUACA Cel-miR-273 SEQ ID NO. 250 SEQ ID NO. 556 UGCCCGUACUGUGUCGGCUGCU AGCAGCCGACACAGUACGGGCA

TABLE 4 Drosophila microRNA and anti-microRNA sequences. microRNA sequence Anti-microRNA molecule microRNA name (5′ to 3′) sequence (5′ to 3′) Dme-miR-263a SEQ ID NO. 251 SEQ ID NO. 557 GUUAAUGGCACUGGAAGAAUUC GAAUUCUUCCAGUGCCAUUAAC Dme-miR-184 SEQ ID NO. 252 SEQ ID NO. 558 UGGACGGAGAACUGAUAAGGGC GCCCUUAUCAGUUCUCCGUCCA Dme-miR-274 SEQ ID NO. 253 SEQ ID NO. 559 UUUUGUGACCGACACUAACGGG CCCGUUAGUGUCGGUCACAAAA Dme-miR-275 SEQ ID NO. 254 SEQ ID NO. 560 UCAGGUACCUGAAGUAGCGCGC GCGCGCUACUUCAGGUACCUGA Dme-miR-92a SEQ ID NO. 255 SEQ ID NO. 561 CAUUGCACUUGUCCCGGCCUAU AUAGGCCGGGACAAGUGCAAUG Dme-miR-219 SEQ ID NO. 256 SEQ ID NO. 562 UGAUUGUCCAAACGCAAUUCUU AAGAAUUGCGUUUGGACAAUCA Dme-miR-276a SEQ ID NO. 257 SEQ ID NO. 563 UAGGAACUUCAUACCGUGCUCU AGAGCACGGUAUGAAGUUCCUA Dme-miR-277 SEQ ID NO. 258 SEQ ID NO. 564 UAAAUGCACUAUCUGGUACGAC GUCGUACCAGAUAGUGCAUUUA Dme-miR-278 SEQ ID NO. 259 SEQ ID NO. 565 UCGGUGGGACUUUCGUCCGUUU AAACGGACGAAAGUCCCACCGA Dme-miR-133 SEQ ID NO. 260 SEQ ID NO. 566 UUGGUCCCCUUCAACCAGCUGU ACAGCUGGUUGAAGGGGACCAA Dme-miR-279 SEQ ID NO. 261 SEQ ID NO. 567 UGACUAGAUCCACACUCAUUAA UUAAUGAGUGUGGAUCUAGUCA Dme-miR-33 SEQ ID NO. 262 SEQ ID NO. 568 AGGUGCAUUGUAGUCGCAUUGU ACAAUGCGACUACAAUGCACCU Dme-miR-280 SEQ ID NO. 263 SEQ ID NO. 569 UGUAUUUACGUUGCAUAUGAAA UUUCAUAUGCAACGUAAAUACA Dme-miR-281 SEQ ID NO. 264 SEQ ID NO. 570 UGUCAUGGAAUUGCUCUCUUUG CAAAGAGAGCAAUUCCAUGACA Dme-miR-282 SEQ ID NO. 265 SEQ ID NO. 571 AAUCUAGCCUCUACUAGGCUUU AAAGCCUAGUAGAGGCUAGAUU Dme-miR-283 SEQ ID NO. 266 SEQ ID NO. 572 UAAAUAUCAGCUGGUAAUUCUG CAGAAUUACCAGCUGAUAUUUA Dme-miR-284 SEQ ID NO. 267 SEQ ID NO. 573 UGAAGUCAGCAACUUGAUUCCA UGGAAUCAAGUUGCUGACUUCA Dme-miR-34 SEQ ID NO. 268 SEQ ID NO. 574 UGGCAGUGUGGUUAGCUGGUUG CAACCAGCUAACCACACUGCCA Dme-miR-124 SEQ ID NO. 269 SEQ ID NO. 575 UAAGGCACGCGGUGAAUGCCAA UUGGCAUUCACCGCGUGCCUUA Dme-miR-79 SEQ ID NO. 270 SEQ ID NO. 576 UAAAGCUAGAUUACCAAAGCAU AUGCUUUGGUAAUCUAGCUUUA Dme-miR-276b SEQ ID NO. 271 SEQ ID NO. 577 UAGGAACUUAAUACCGUGCUCU AGAGCACGGUAUUAAGUUCCUA Dme-miR-210 SEQ ID NO. 272 SEQ ID NO. 578 UUGUGCGUGUGACAGCGGCUAU AUAGCCGCUGUCACACGCACAA Dme-miR-285 SEQ ID NO. 273 SEQ ID NO. 579 UAGCACCAUUCGAAAUCAGUGC GCACUGAUUUCGAAUGGUGCUA Dme-miR-100 SEQ ID NO. 274 SEQ ID NO. 580 AACCCGUAAAUCCGAACUUGUG CACAAGUUCGGAUUUACGGGUU Dme-miR-92b SEQ ID NO. 275 SEQ ID NO. 581 AAUUGCACUAGUCCCGGCCUGC GCAGGCCGGGACUAGUGCAAUU Dme-miR-286 SEQ ID NO. 276 SEQ ID NO. 582 UGACUAGACCGAACACUCGUGC GCACGAGUGUUCGGUCUAGUCA Dme-miR-287 SEQ ID NO. 277 SEQ ID NO. 583 UGUGUUGAAAAUCGUUUGCACG CGUGCAAACGAUUUUCAACACA Dme-miR-87 SEQ ID NO. 278 SEQ ID NO. 584 UUGAGCAAAAUUUCAGGUGUGU ACACACCUGAAAUUUUGCUCAA Dme-miR-263b SEQ ID NO. 279 SEQ ID NO. 585 CUUGGCACUGGGAGAAUUCACA UGUGAAUUCUCCCAGUGCCAAG Dme-miR-288 SEQ ID NO. 280 SEQ ID NO. 586 UUUCAUGUCGAUUUCAUUUCAU AUGAAAUGAAAUCGACAUGAAA Dme-miR-289 SEQ ID NO. 281 SEQ ID NO. 587 UAAAUAUUUAAGUGGAGCCUGC GCAGGCUCCACUUAAAUAUUUA Dme-bantam SEQ ID NO. 282 SEQ ID NO. 588 UGAGAUCAUUUUGAAAGCUGAU AUCAGCUUUCAAAAUGAUCUCA Dme-miR-303 SEQ ID NO. 283 SEQ ID NO. 589 UUUAGGUUUCACAGGAAACUGG CCAGUUUCCUGUGAAACCUAAA Dme-miR-31b SEQ ID NO. 284 SEQ ID NO. 590 UGGCAAGAUGUCGGAAUAGCUG CAGCUAUUCCGACAUCUUGCCA Dme-miR-304 SEQ ID NO. 285 SEQ ID NO. 591 UAAUCUCAAUUUGUAAAUGUGA UCACAUUUACAAAUUGAGAUUA Dme-miR-305 SEQ ID NO. 286 SEQ ID NO. 592 AUUGUACUUCAUCAGGUGCUCU AGAGCACCUGAUGAAGUACAAU Dme-miR-9c SEQ ID NO. 287 SEQ ID NO. 593 UCUUUGGUAUUCUAGCUGUAGA UCUACAGCUAGAAUACCAAAGA Dme-miR-306 SEQ ID NO. 288 SEQ ID NO. 594 UCAGGUACUUAGUGACUCUCAA UUGAGAGUCACUAAGUACCUGA Dme-miR-9b SEQ ID NO. 289 SEQ ID NO. 595 UCUUUGGUGAUUUUAGCUGUAU AUACAGCUAAAAUCACCAAAGA 0 Dme-miR-125 SEQ ID NO. 290 SEQ ID NO. 596 UCCCUGAGACCCUAACUUGUGA UCACAAGUUAGGGUCUCAGGGA Dme-miR-307 SEQ ID NO. 291 SEQ ID NO. 597 UCACAACCUCCUUGAGUGAGCG CGCUCACUCAAGGAGGUUGUGA Dme-miR-308 SEQ ID NO. 292 SEQ ID NO. 598 AAUCACAGGAUUAUACUGUGAG CUCACAGUAUAAUCCUGUGAUU dme-miR-31a SEQ ID NO. 293 SEQ ID NO. 599 UGGCAAGAUGUCGGCAUAGCUG CAGCUAUGCCGACAUCUUGCCA dme-miR-309 SEQ ID NO. 294 SEQ ID NO. 600 GCACUGGGUAAAGUUUGUCCUA UAGGACAAACUUUACCCAGUGC dme-miR-310 SEQ ID NO. 295 SEQ ID NO. 601 UAUUGCACACUUCCCGGCCUUU AAAGGCCGGGAAGUGUGCAAUA dme-miR-311 SEQ ID NO. 296 SEQ ID NO. 602 UAUUGCACAUUCACCGGCCUGA UCAGGCCGGUGAAUGUGCAAUA dme-miR-312 SEQ ID NO. 297 SEQ ID NO. 603 UAUUGCACuUGAGACGGCCUGA UCAGGCCGUCUCAAGUGCAAUA dme-miR-313 SEQ ID NO. 298 SEQ ID NO. 604 UAUUGCACUUUUCACAGCCCGA UCGGGCUGUGAAAAGUGCAAUA dme-miR-314 SEQ ID NO. 299 SEQ ID NO. 605 UAUUCGAGCCAAUAAGUUCGG CCGAACUUAUUGGCUCGAAUA dme-miR-315 SEQ ID NO. 300 SEQ ID NO. 606 UUUUGAUUGUUGCUCAGAAAGC GCUUUCUGAGCAACAAUCAAAA dme-miR-316 SEQ ID NO. 301 SEQ ID NO. 607 UGUCUUUUUCCGCUUACUGGCG CGCCAGUAAGCGGAAAAAGACA dme-miR-317 SEQ ID NO. 302 SEQ ID NO. 608 UGAACACAGCUGGUGGUAUCCA UGGAUACCACCAGCUGUGUUCA dme-miR-318 SEQ ID NO. 303 SEQ ID NO. 609 UCACUGGGCUUUGUUUAUCUCA UGAGAUAAACAAAGCCCAGUGA dme-miR-2c SEQ ID NO. 304 SEQ ID NO. 610 UAUCACAGCCAGCUUUGAUGGG CCCAUCAAAGCUGGCUGUGAUA Dme-miR-iab45p SEQ ID NO. 305 SEQ ID NO. 611 ACGUAUACUGAAUGUAUCCUGA UCAGGAUACAUUCAGUAUACGU Dme-miR-iab43p SEQ ID NO. 306 SEQ ID NO. 612 CGGUAUACCUUCAGUAUACGUA UACGUAUACUGAAGGUAUACCG

EXAMPLES Example 1 Materials and Methods

Oligonucleotide Synthesis

MiR-21 were synthesized using 5′-silyl, 2′-ACE phosphoramidites (Dharmacon, Lafayette, Colo., USA) on 0.2 μmol synthesis columns using a modified ABI 394 synthesizer (Foster City, Calif., USA) (Scaringe, Methods Enzymol. 317, 3-18 (2001) and Scaringe, Methods 23, 206-217 (2001)). The phosphate methyl group was removed by flushing the column with 2 ml of 0.2 M 2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate in DMF/water (98:2 v/v) for 30 min at room temperature. The reagent was removed and the column rinsed with 10 ml water followed by 10 ml acetonitrile. The oligonucleotide was cleaved and eluted from the solid support by flushing with 1.6 ml of 40% aqueous methylamine over 2 min, collected in a screwcap vial and incubated for 10 min at 55° C. Subsequently, the base-treated oligonucleotide was dried down in an Eppendorf concentrator to remove methylamine and water. The residue was dissolved -in sterile 2′-deprotection buffer (400 μl of 100 mM acetate-TEMED, pH3.8, for a 0.2 μmol scale synthesis) and incubated for 30 minutes at 60° C. to remove the 2′ ACE group. The oligoribonucleotide was precipitated from the acetate-TEMED solution by adding 24 μl 5 M NaCl and 1.2 ml of absolute ethanol.

2′-O-Methyl oligoribonucleotides were synthesized using 5′-DMT, 2′-O-methyl phosphoramidites (Proligo, Hamburg, Germany) on 1 μmol synthesis columns loaded with 3′-aminomodifier (TFA) C7 Icaa control pore glass support (Chemgenes, Mass., USA). The aminolinker was added in order to also use the oligonucleotides for conjugation to amino group reactive reagents, such as biotin succinimidyl esters. The synthesis products were deprotected for 16 h at 55° C. in 30% aqueous ammonia and then precipitated by the addition of 12 ml absolute 1-butanol. The fall-length product was then gel-purified using a denaturing 20% polyacrylamide gel. 2′-Deoxyoligonucleotides were prepared using 0.2 μmol scale synthesis and standard DNA synthesis reagents (Proligo, Hamburg, Germany).

The sequences of the 2′-O-methyl oligoribonucleotides were 5′-GUCAACAUCAGUCUGAUAAGCUAL (L, 3′ aminolinker) for 2′-OMe miR-21(SEQ ID NO. 613), and 5′-AAGGCAAGCUGACCCUGAAGUL for EGFP 2′-OMe antisense (SEQ ID NO. 614), 5′-UGAAGUCCCAGUCGAACGGAAL for EGFP 2′-OMe reverse (SEQ ID NO. 615); the sequence of chimeric 2′-OMe/DNA oligonucleotides was 5′-GTCAACATCAGTCTGATAAGCTAGCGL for 2′-deoxy miR-21(underlined, 2′-OMe residues)(SEQ ID NO. 616), and 5′-AAGGCAAGCTGACCCTGAAGTGCGL for EGFP 2′-deoxy antisense (SEQ ID NO. 617).

The miR-21 cleavage substrate was prepared by PCR-based extension of the partially complementary synthetic DNA oligonucleotides 5′-GAACAATTGCTTTTACAGATGCACATATCGAGGTGAACATCACGTACGTCAACATCAGTCTGATA AGCTATCGGTTGGCAGAAGCTAT (SEQ ID NO. 618) and 5′-GGCATAAAGAATTGAAGAGAGTTTTCACTGCATACGACGATTCTGTGATTTGTATTCAGCCCATAT CGTTTCATAGCTTCTGCCAACCGA (SEQ ID NO. 619). The extended dsDNA was then used as template for a new PCR with primers 5′-TAATACGACTCACTATAGAACAATTGCTTTTACAG (SEQ ID NO. 620) and 5′-ATTTAGGTGACACTATAGGCATAAAGAATTGAAGA (SEQ ID NO. 621) to introduce the T7 and SP6 promoter sequences for in vitro transcription. The PCR product was ligated into pCR2.1-TOPO (Invitrogen). Plasmids isolated from sequence-verified clones were used as templates for PCR to produce sufficient template for run-off in vitro transcription reactions using phage RNA polymerases (Elbashir et al., EMBO 20, 6877-6888(2001)). ³²P-Cap-labelling was performed as reported (Martinez et al., Cell 110, 563-574(2002)).

Plasmids

Plasmids pEGFP-S-21 and pEGFP-A-21 were generated by T4 DNA ligation of preannealed oligodeoxynucleotides 5′-GGCCTCAACATCAGTCTGATAAGCTAGGTACCT (SEQ ID NO. 622) and 5′-GGCCAGGTACCTAGCTTATCAGACTGATGTTGA (SEQ ID NO. 623)into NotI digested pEGFP-N-1(Clontech). The plasmid pHcRed-C1 was from Clontech.

HeLa Extracts and miR-21 Quantification

HeLa cell extracts were prepared as described (Dignam et al., Nucleic Acid Res. 11 1475-1489 (1983)). 5×10⁹ cells from HeLa suspension cultures were collected by centrifugation and washed with PBS (pH7.4). The cell pellet (approx. 15 ml) was re-suspended in two times of its volume with 10 mM KCl/1.5 mM MgCl₂/0.5 mM dithiothreitol/10 mM HEPES-KOH (pH 7.9) and homogenized by douncing. The nuclei were then removed by centrifugation of the cell lysate at 1000 g for 10 min. The supernatant was spun in an ultracentrifuge for 1 h at 10,5000 g to obtain the cytoplasmic S100 extract. The concentration of KCl of the S100 extract was subsequently raised to 100 mM by the addition of 1 M KCl. The extract was then supplemented with 10% glycerol and frozen in liquid nitrogen.

280 μg of total RNA was isolated from 1 ml of S100 extract using the acidic guanidinium thiocyanate-phenol-chloroform extraction method (Chomczynski et al., Anal. Biochem. 162, 156-159 (1987)). A calibration curve for miR-21 Northern signals was produced by loading increasing amounts (10 to 30000 pg) of synthetically made miR-21 (Lim et al. et al., Genes & Devel. 17, 991-1008 (2003)). Northern blot analysis was performed as described using 30 μg of total RNA per well (Lagos-Quintana et al., Science 294, 853-858 (2001)).

In vitro miRNA Cleavage and Inhibition Assay

2′-O-Methyl oligoribonucleotides or 2′-deoxyoligonucleotides were pre-incubated with HeLa S100 at 30° C. for 20 min prior to the addition of the cap-labeled miR-21 target RNA. The concentration of the reaction components were 5 nM target RNA, 1 mM ATP, 0.2 mM GTP, 10 U/ml RNasin (Promega) and 50% HeLa S100 extract in a final reaction volume of 25 μl. The reaction time was 1.5 h at 30° C. The reaction was stopped by addition of 200 μl of 300 mM NaCl/25 mM EDTA/20% w/v SDS/200 mM Tris HCl (pH7.5). Subsequently, proteinase K was added to a final concentration of 0.6 mg/ml and the sample was incubated for 15 min at 65° C. After phenol/chloroform extraction, the-RNA was ethanol-precipitated and separated on a 6% denaturing polyacrylamide gel. Radioactivity was detected by phosphorimaging.

Cell Culture and Transfection

HeLa S3 and HeLa S3/GFP were grown in 5% CO2 at 37° C. in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 unit/ml penicillin, and 100 μg/ml streptomycin. One day before transfection, 105 cells were plated in 500 μl DMEM containing 10% FBS per well of a 24-well plate. Plasmid and plasmid/oligonucleotide transfection was carried out with Lipofectamine2000 (Invitrogen). 0.2 μg pEGFP or its derivatives were cotransfected with 0.3 μg pHcRed with or without 10 pmol of 2′-O-methyl oligoribonucleotide or 10 pmol of 2′-deoxyoligonucleotide per well. Fluorescent cell images were recorded on a Zeiss Axiovert 200 inverted fluorescence microscope (Plan-Apochromat 10×0.45) equipped with Chroma Technology Corp. filter sets 41001 (EGFP) and 41002c (HcRed) and AxioVision 3.1 software.

Example 2 MicroRNA-21 Cleavage of Target RNA

In order to assess the ability of modified oligonucleotides to specifically interfere with miRNA function, we used our previously described mammalian biochemical system developed for assaying RISC activity (Martinez et al., Cell 100, 563-574 (2002)). Zamore and colleagues (Hutvágner et al., Science 297, 2056-2050 (2002)) showed that crude cytoplasmic cell lysates and eIF2C2 immunoprecipitates prepared from these lysates contain let-7 RNPs that specifically cleave let-7-complementary target RNAs. We previously reported that in HeLa cells, numerous miRNAs are expressed including several let-7 miRNA variants (Lagos-Quintana et al., Science 294, 853-858 (2001)).

To assess if other HeLa cell miRNAs are also engaged in RISC like miRNPs we examined the cleavage of a 32P-cap-labelled substrate RNA with a complementary site to the highly expressed miR-21 (Lagos-Quintana et al., Science 294, 853-858 (2001); Mourelatos et al., Genes & Dev. 16, 720-728 (2002)). Sequence-specific target RNA degradation was readily observed and appeared to be approximately 2- to 5-fold more effective than cleavage of a similar let-7 target RNA (FIG. 2A, lane 1, and data not shown). We therefore decided to interfere with miR-21 guided target RNA cleavage.

Example 3 Anti MicroRNA-21 2′-O-methyl Oligoribonucleotide Inhibited MicroRNA-21-Induced Cleavage of Target RNA

A 24-nucleotide 2′-O-methyl oligoribonucleotide that contained a 3′ C7 aminolinker and was complementary to the longest form of the miR-21 was synthesized. The aminolinker was introduced in order to enable post-synthetic conjugation of non-nucleotidic residues such as biotin.

Increasing concentrations of anti miR-21 2′-O-methyl oligoribonucleotide and a control 2′-O-methyl oligoribonucleotide cognate to an EGFP sequence were added to the S100 extract 20 min prior to the addition of 32P-cap-labelled substrate. We determined the concentration of miR-21 in the S100 extract by quantitative Northern blotting to be 50 pM (Lim et al., Genes & Devel. 17,-991-1008 (2003)).

The control EGFP oligonucleotide did not interfere with miR-21 cleavage even at the highest applied concentration (FIG. 2A, lanes 2-3). In contrast, the activity of miR-21 was completely blocked at a concentration of only 3 nM (FIG. 2A, lane 5), and a concentration of 0.3 nM showed a substantial 60%-70% reduction of cleavage activity (FIG. 2, lane 6) At a concentration of 0.03 nM, the cleavage activity of miR-21 was not affected when compared to the lysate alone (FIG. 2, lane 1,7).

Antisense 2′-deoxyoligonucleotides (approximately 90% DNA molecules) at concentrations identical to those of 2′-O-methyl oligoribonucleotides, we could not detect blockage of miR-21 induced cleavage (FIG. 2A, lanes 8-10). The 2′-deoxynucleotides used in this study were protected-against 3′-exonucleases by the addition of three 2′-O-methyl ribonucleotide residues.

Example 4 Anti MicroRNA-21 2′-O-methyl Oligoribonucleotide Inhibited MicroRNA-21-Induced Cleavage of Target RNA In Vitro

In order to monitor the activity of miR-21 in HeLa cells, we constructed reporter plasmids that express EGFP mRNA that contains in its 3′ UTR a 22-nt sequence complementary to miR-21 (pEGFP-S-21) or in sense orientation to miR-21 (p-EGFP-A-21). Endogenous miRNAs have previously been shown to act like siRNAs by cleaving reporter mRNAs carrying sequences perfectly complementary to miRNA. To monitor transfection efficiency and specific interference with the EGFP indicator plasmids, the far-red fluorescent protein encoding plasmid pHcRed-C1 was cotransfected.

Expression of EGFP was observed in HeLa cells transfected with pEGFP and pEGFP-A-21 (FIG. 3, rows 1 and 2), but not from those transfected with pEGFP-S-21 (FIG. 3, row 3). However, expression of EGFP from pEGFP-S-21 was restored upon cotransfection with anti miR-21 2′-O-methyl oligoribonucleotide (FIG. 3, row 4). Consistent with our above observation, the 2′-deoxy anti miR-21 oligonucleotide showed no effect (FIG. 3, row 5). Similarly, cotransfection of the EGFP 2′-O-methyl oligoribonucleotide in sense orientation with respect to the EGFP mRNA (or antisense to EGFP guide siRNA) had no effect (FIG. 3,row 6).

We have demonstrated that miRNP complexes can be effectively and sequence-specifically inhibited with 2′-O-methyl oligoribonucleotides antisense to the guide strand positioned in the RNA silencing complex. 

1. An isolated molecule comprising a maximum of fifty moieties, wherein each moiety comprises a base bonded to a backbone unit said molecule comprising the microRNA molecule identified in SEQ ID NO: 139 or its corresponding anti-micro RNA molecule identified in SEQ ID NO:
 445. 2. A molecule according to claim 1, wherein the molecule is modified for increased nuclease resistance.
 3. The molecule according to claim 1, wherein at least one of the moieties is a modified ribonucleotide moiety.
 4. The molecule according to claim 3, wherein the modified ribonucleotide is substituted at the 2′ position.
 5. The molecule according to claim 4, wherein the substituent at the 2′ position is a C₁ to C₄ alkyl group.
 6. The molecule according to claim 5, wherein the alkyl group is methyl.
 7. The molecule according to claim 5, wherein the alkyl group is allyl.
 8. The molecule according to claim 4, wherein the substituent at the 2′ position is a C₁ to C₄ alkoxy-C₁ to C₄ alkyl group.
 9. The molecule according to claim 8, wherein the C₁ to C₄ alkoxy-C₁ to C₄ alkyl group is methoxyethyl.
 10. The molecule according to claim 1, wherein at least one of the moieties is a 2′-fluororibonucleotide moiety.
 11. The molecule according to claim 3, wherein the modified ribonucleotide has a methylene bridge between the 2′-oxygen atom and the 4′-carbon atom.
 12. The molecule according to claim 1, wherein the molecule comprises at least one modified moiety on the 5′ end.
 13. The molecule according to claim 1, wherein the molecule comprises at least two modified moieties at the 5′ end.
 14. The molecule according to claim 1, wherein the molecule comprises at least one modified moiety on the 3′ end.
 15. The molecule according to claim 1, wherein the molecule comprises at least two modified moieties at the 3′ end.
 16. The molecule according to claim 1, wherein the molecule comprises at least two modified moieties at the 5′ end and at least two modified moieties at the 3′ end.
 17. The molecule according to claim 1, wherein the molecule comprises a nucleotide cap at the 5′ end, the 3′ end or both.
 18. The molecule according to claim 1, wherein the molecule consists of the microRNA molecule identified in SEQ ID NO:
 139. 19. The molecule according to claim 1, wherein the molecule consists of the anti-micro RNA molecule identified in SEQ ID NO:
 445. 